Rare diseases (RD) are characterized by low prevalence and affect not more than five individuals per 10,000 in the European population; they are a large and heterogeneous group of disorders including more than 7,000 conditions and often involve all organs and tissues, with several clinical subtypes within the same disease. Very often information concerning either diagnosis and/or prognosis on many RD is insufficient. microRNAs are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level by either degrading or blocking translation of messenger RNA targets. Recently, microRNA expression patterns of body fluids underscored their potential as noninvasive biomarkers for various diseases. The role of microRNAs as potential biomarkers has become particularly attractive. The identification of disease-related microRNAs is essential for understanding the pathogenesis of diseases at the molecular level, and is critical for designing specific molecular tools for diagnosis, treatment and prevention. Computational analysis of microRNA-disease associations is an important complementary means for prioritizing microRNAs for further experimental examination. In this article, we explored the added value of miRs as biomarkers in a selected panel of RD hitting different tissues/systems at different life stages, but sharing the need of better biomarkers for diagnostic and prognostic purposes. According to the Regulation EC n.141/2000, rare diseases (RD) are defined on the basis of low prevalence and affect not more than five individuals per 10,000 in the European population [In the USA the Orphan Drug Act of 1983 defines a RD as “any disease or condition that affects less than 200,000 people in the United States”. In Japan, the Japanese Medicines Act of 1993 defines a rare disease as a condition affecting no more than 50,000 people in the country.They are a large and diverse group of disorders, estimated to include more than 6000 conditions, often involving all organs and tissues with several clinical subtypes within the same disease. Several groups of RD include congenital malformations, metabolic defects, rare tumors, skin diseases, syndromes involving body systems such as the nervous, immune, excretory, reproductive, cutaneous ones. About 80% of RD have genetic origins, involving one or several genes or chromosomal abnormalities; in many cases the clinical phenotype and prognosis appear to be modulated by yet unknown factors, such as different mutations in the same gene(s) or also epigenetic factors.The difficulty in obtaining the correct diagnosis is the first dramatic hurdle for rare disease patients. It has been estimated that about 30% of RD are still lacking a diagnostic definition; moreover, many RD cases get an accurate diagnosis late, even after five years or more from disease recognition. Late diagnoses delay specific treatments and might have severe, irreversible, debilitating and life-threatening consequences .etc.), information on many RD are insufficient concerning either diagnosis and/or prognosis.Accurate and timely diagnosis is often of principal importance for prevention and properly targeted treatment; where these are unavailable, timely diagnosis is always important for protecting the patients’ quality of life. However, due to problems associated with rarity  has been formed under the auspices of the US National Institutes of Health (NIH) and the European Commission (EC) with the ambitious goal of developing a diagnostic tool for every known rare disease by 2020, along with new therapies to treat 200 of them [Furthermore, it is now well ascertained that RD suffer from the relative lack of information from natural history studies of diseases. The specificities of rare diseases—limited number of patients and scarcity of relevant knowledge and expertise—can be tackled through a global approach. Very recently of them .microRNAs (miRs) are regulated and transcribed like protein coding genes. Subsequent miR biogenesis involves discrete processing and transport steps, whereby the active moiety of 20–22 nucleotides is excised from a longer RNA precursor that exhibits specific hairpin structure. Finally, these 20–22 nucleotides are incorporated into a composite machinery, which promotes partial duplex formation between the short RNA and the untranslated regions (UTR) of targeted mRNAs, resulting typically in translational silencing in mammals.Although the total number of miR remains controversial and their roles are only beginning to be defined, miR expression analyses indicate that diverse tumors display miRs expression profiles (for mature and/or precursor miRs) significantly different from normal tissue. Furthermore, miRs are emerging as highly tissue-specific biomarkers with potential clinical applicability for defining the cancer origin of metastases. They have been appreciated in various fundamental biological processes such as cell proliferation , stem ceFuture research targets integrate sets of new biomarkers to be developed with a system biology approach and in combination with bioinformatics tools, to devise powerful diagnostic and prognostic algorithms. Ideally, biomarkers should be easily accessible such that they can be sampled non-invasively. Therefore biomarkers that can be sampled from body fluids, such as serum or urine, are particularly desirable. In recent years it has become clear that miRs could represent new effective biomarkers.miRs as a diagnostic tool might be used to detect an increased risk of acquiring RD and cancer by studying disease associated variations of miR sequences (like miR-146a in thyroid cancer) ; to diagUsefulness of miRs as new effective biomarkers is due to the fact that (i) miRs expression is known to be aberrant in cancer; (ii) miRs expression profiles are pathognomonic, or tissue-specific; (iii) over expression or lack of expression of specific miRs appear to correlate with clinically aggressive or metastatic phenotypes; (iv) miRs are remarkably stable molecules being well preserved in formalin fixed, paraffin embedded tissues, fresh snap frozen specimens and body fluids.Very few studies have been reported on possible alternative mechanisms underlying specific RDs onset and clinical manifestations and progression. This causes the lack of diagnostic/prognostic markers that are strongly needed for an adequate follow-up of patients, currently based only on classical screenings.The identification of miRs associated with human diseases is an important goal of biomedical research. Recently, a number of computational methods have been developed to predict or prioritize diseases-related genes. Most approaches are based on the idea that dysfunctions of functionally related genes tend to be associated with phenotypically similar diseases. These genes linked to similar diseases usually interact with each other or participate in the common biological modules. Network-based approaches have also been employed to predict or prioritize new candidate disease genes based upon network linkages with known disease genes. These approaches typically start with constructing a gene-gene association network based on one or more types of genomic and proteomic information, and then prioritize candidate protein-coding genes based on network proximity to known disease-related genes .Even though few studies are available on miRs and their association with RDs, some evidence is emerging on their role in controlling pathways involved in RDs onset and/or progression. In this paragraph we briefly review the latest findings on selected RDs hitting different tissues/systems at different life stages, but sharing the need of better biomarkers for diagnostic and prognostic purposes .Duchenne muscular dystrophy  is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. It has been recently demonstrated that the serum levels of muscle-specific miRs  are released in to the bloodstream of DMD patients [patients ; increaspatients . Recent patients –21. Morepatients , DMD, anpatients ,24. Yuaspatients  showed tpatients  or that Amyotrophic lateral sclerosis  is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Williams and colleagues showed that a key regulator of this RD is the miR-206, a skeletal muscle-specific miR that is dramatically induced in a mouse model of ALS. Mice that are genetically deficient in miR-206 form normal neuromuscular synapses during development, but deficiency of miR-206 in the ALS mouse model accelerates disease progression. MiR-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury, which probably accounts for its salutary effects in ALS [s in ALS .Sezary syndrome (SS) is a rare and aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) characterized by the presence of neoplastic lymphocytes named Sezary cells, in the skin, lymph nodes and peripheral blood [al blood . Expressal blood . Anotheral blood . van deral blood .Rett syndrome  is a complex neurological disorder that has been associated with mutations in the gene coding for the methyl CpG binding protein 2 (Mecp2). This X-linked neurological disorder causes a severe phenotype in one out of every 10,000–15,000 live births, making it the second most frequent cause of mental retardation in females. Very recently it has been examined the possible effects of miR misregulation caused by Mecp2 absence in a mouse model of RTT. Using miR expression microarrays, it was observed that the brain of Rett syndrome mice undergoes a disruption of the expression profiles of miRs. Among the significantly altered miRs , overall downregulation of these transcripts was the most common feature (71%), while the remaining 30% were upregulated. In particular it was demonstrated that the most commonly disrupted miRs were miR-146a, miR-146b, miR-130, miR-122a, miR-342 and miR-409 (downregulated) and miR-29b, miR329, miR-199b, miR-382, miR-296, miR-221 and miR-92 (upregulated) [Multiple osteochondromas  are characterized by a large spectrum of germline mutations scattered along EXT1/EXT2 genes, by the presence of a significant percentage of patients without alterations in the EXT genes and a large phenotypic intra- and interfamilial variability. The molecular basis of MOs genetic and clinical heterogeneity are currently unknown. This leads to the lack of appropriate diagnostic/prognostic markers as well as of therapeutic options for both benign and malignant lesions. 45 miRs have been identified as differentially expressed in MO patients, and classified them into 4 classes according to their expression compared to articular and/or growth plate cartilage. In particular a signature of 8 miRs  was able to distinguish healthy growth control from MO patients. Compared to microarray data clustering, this finding allows to more accurately identify miRs specifically related to pathologic condition of growth plate cartilage. Results indicate that miRs differentially expressed may hamper the molecular signaling responsible for normal proliferation and differentiation of chondrocytes, thus contributing to pathogenesis and clinical outcome [ outcome .Hailey-Hailey disease  is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation leading to the development of erosive and oozing skin lesions. While a strong relationship exists between mutations in the gene ATP2C1 and HHD, a poor understanding of how these mutations affect manifestations of the disease is available. A specific signature discriminating lesion from normal skin was recently found. The authors found, that miR-181a, miR-125b, and miR-99a expression increases in HHD-lesion derived keratinocytes. Interestingly, the expression of miR-181a, miR-125b and miR-99a has an anti-proliferative effect in several cell types [ll types –35. Addill types  was founll types ,38. MiR-ll types .Hepatoblastoma  is a rare pediatric liver tumor with an incidence in Western countries of 1.5 cases per million of individuals younger than 15 years. Despite this, it is the most common hepatic malignancy during childhood. Although specific chromosome aberrations have not been linked with prognosis or casual factors, genetic disorders have been described in chromosomes 1q, 2q, 7q, 8, 17q and 20 as well as activation of Wnt signaling pathway through mutations in its central mediator beta-catenin. Whole genome analysis of miRs showed that 4 miRs were significantly upregulated in the tumor compared with the nonmalignant tissue  and one was significantly downregulated (miR-148a). Among these, miR-214 has been shown to be deeply involved in controlling the level of PTEN protein, a negative regulator of the PI3K signaling whose alteration has been shown to give rise to different cancers [The  cancers . Finally cancers .The use of miRs as biomarkers is a promising field. Their basic biology is still being elucidated and questions regarding tissue-specific functions and targets, developmental time of expression, regulation and evolutionary role are being explored. With their key roles in gene expression regulation, particularly during cell differentiation and tissue genesis, miRs are probably critical in cancer initiation and progression, but more research studies are needed to explore their impact as clinical biomarkers for cancer and disease diagnosis, prognosis and prediction of therapeutic response. Outputs of this new field will be the identification of molecular markers to detect potential therapeutic targets, new biomarkers which could improve diagnosis and management, optimize patients’ follow-up and, in turn, improve their quality of life. The development of minimally invasive tests for the detection and monitoring of RD can reduce their worldwide health burden. Although conventional strategies for blood-based biomarker discovery  have shown promise, the development of clinically validated cancer detection markers remains an unmet challenge for many common human cancers. It is strongly believed that studies on the biological basis of the above mentioned pathologies will improve their understanding and support the development of perspective therapies. The identification of others disease-related miRs is extremely important for understanding the pathogenesis of diseases at the molecular level and is critical for designing specific molecular tools for diagnosis, treatment and prevention.More than 1500 transcribed miRs have been identified in the human genome since their discovery. miR analysis is a promising tool for identifying new biomarkers, as well as for predicting response to cancer and rare diseases therapy. Recently, several reports suggest that cell-free circulating miRs are detectable in serum/plasma and the levels of tumor-derived miRs are altered in patients with tongue cancer, lung cancer, prostate cancer, ovarian cancer, and colorectal cancer (reviewed in this issue). These findings suggest that blood-based miRs could emerge as revolutionary sources of biomarker for diagnosis. Further understanding of their functional roles will open up new opportunities in developing revolutionary therapeutic strategies. Although we are just beginning to understand the specific contribution of miRs in RD, a larger sample sets including long-term clinical data are urgently required for future studies. Finally, major efforts need to be applied on standardization of isolation, quantification and normalization strategies before any of the novel miR biomarkers is applicable for clinical practice.


Idiopathic pulmonary fibrosis (IPF), the most common of the idiopathic interstitial pneumonias, is a devastating condition that carries a prognosis worse than that of many cancers. As such, it represents one of the most challenging diseases for chest physicians. The diagnostic process is complex and relies on the clinician integrating clinical, laboratory, radiologic, and/or pathologic data. Therefore, a close collaboration between chest physicians, radiologists, and pathologists experienced in the diagnosis of interstitial lung diseases (ILDs) is necessary in order to minimize diagnostic uncertainty. Similarly, the management of IPF continues to pose major difficulties. However, while there are no proven effective therapies for IPF beyond lung transplantation, recent trials of novel agents suggest that pharmacological treatment may retard the progression of the disease. In this regard, enrolment of patients into clinical trials is considered the “best current practice”by the most recent guidelines as it offers IPF patients the chance to receive new agents that may be more effective than current therapies. A more recent trend focusing on improving quality of life in IPF patients has also been gaining ground.The diagnosis and management of IPF remains a constant challenge for even the most experienced of clinicians. However, a multidisciplinary approach to this complex disease is steadily improving diagnostic accuracy, while recent advances in the pharmacological therapy offer the genuine promise of future treatments for this devastating disease. Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown cause, limited to the lung and associated with the histopathological  and/or radiological  pattern of usual interstitial pneumonia (UIP) . The disDiagnosing IPF in daily practice may be challenging and there is often a significant delay between the first manifestations of the disease - typically a combination of dyspnea on exertion and dry cough - and its correct diagnosis . MoreoveThis review focuses on current diagnostic and therapeutic challenges in IPF. Timely diagnosis and early referral of patients to centers with specific expertise may lead to more optimal disease management.Suspicion of IPF often arises several months after the initial manifestations of the disease. In fact, dry cough and exertional dyspnea - the most common presenting symptoms - tend to be overlooked and wrongly attributed to smoking habits or aging. Uncertainties, thus diagnostic delay, may be due to a number of reasons. Sometimes, patients may be too sick to undergo surgical, although relatively non-invasive, procedures and this may preclude obtaining tissue samples, which are critical for the final diagnosis in a sizeable proportion of cases. Conversely, in community centers the diagnostic delay could be due to the radiologist or pathologist insufficient experience with ILD. Nevertheless, although the overall accuracy of a diagnosis of IPF in expert centers has been reported to be good (87%), even in this setting the level of agreement within experts is fair to moderate . SignifiBeing the disease “idiopathic” by definition, the diagnosis of IPF requires the exclusion of all known causes of ILD. Because the number of potential “mimickers” of IPF is high, this step is crucial.Familial pulmonary fibrosis (FPF), the occurrence of IPF in two or more members of the same family, accounts for less than 5% of total IPF cases ,15. FamiPatients with ILD should be thoroughly investigated for possible HP since chronic forms - commonly caused by prolonged exposure to low antigen levels - may mimic IPF both clinically and radiologically . BronchoConnective tissue disease (CTD) can present with a UIP pattern  and ILD The differential diagnosis between asbestosis and IPF may not be straightforward owing to the radiological and histopathological similarities between these two entities . Since tWithout an adequate review of a patient’s medical history and past relevant imaging data, fibrotic sarcoidosis  may Table The clinical course of IPF is most commonly characterized by a slowly progressive decline in lung function and worsening dyspnea leading to death within several years of diagnosis . HoweverIn some cases, even at the end of an accurate diagnostic work-up, the nature of the fibrotic process may remain unclear. Possible causes of diagnostic failure could be either technical or atypical manifestations of the disease. For instance, a biopsy taken from the “wrong” area may provide normal lung tissue, indiscernible end-stage lung or a sample that does not meet diagnostic criteria for any specific ILD. In such cases, the biopsy will not be diagnostic.discordant UIP) appear to behave similarly to those with UIP pattern in all lobes (concordant UIP) [In patients whose HRCT does not demonstrate a UIP pattern, a surgical lung biopsy (SLB) is necessary to make a definitive diagnosis. From a technical point of view, the following aspects should be considered: type of surgical approach, side of operation, target areas for the biopsy and number of biopsies. The diagnostic yield from surgical lung biopsies obtained from video-assisted thoracoscopy (VATS) and open thoracotomy are similar, although VATS is associated with lower morbidity and length of stay than open thoracotomy, thus representing the procedure of choice , while aant UIP) ,42. If bant UIP) . The decTable weak recommendation against their use by the most recent guidelines [IPF is a disease with a poor prognosis that appears unchanged over the past decade: the median survival in non-transplanted patients is around 3.5 years after diagnosis ,11. Theridelines ,49. Specslow progressors are less likely to respond to any given drug, thus leading to false negative results.There is a growing appreciation that distinct phenotypes exist within what is currently regarded as IPF. Clinical variants include a temporal spectrum of disease behavior that ranges from a rapid, including acute exacerbations, to a slowly protracted pattern of progression. Interestingly, this latter “survivor” group is equally distributed among disease severity groups . A surviDrugs evaluated to date in IPF have failed to demonstrate an effect on survival . HoweverThere is an increasing awareness of complications and comorbidities frequently associated with IPF  but it iCO), shorter walk distance, desaturation during exercise and increased risk of death [2, DLCO, and quality of life favouring sildenafil [A and ETB receptor antagonist - has also been studied in IPF. Based on a non-predefined subset analysis of a smaller study suggesting that bosentan may prevent disease progression in patients that had undergone a diagnostic surgical lung biopsy [A receptor - has been prematurely stopped following an interim analysis indicating lack of efficacy .Common concomitant conditions include pulmonary hypertension (PH), vascular or coronary artery disease, gastroesophageal reflux disease, obesity, diabetes, emphysema, and obstructive sleep apnoea . PH is cof death . In a reof death . Higher of death . In addiof death . Whetherof death . Howeverldenafil . Bosentag biopsy , a largeg biopsy . SimilarGastroesophageal reflux disease (GERD) is another common comorbidity in patients with IPF , and the likelihood of having both IPF and GERD increases in patients >60 years of age . The preCoronary artery disease (CAD) is more prevalent in IPF patients compared to a similarly matched chronic obstructive pulmonary disease (COPD) group; this association, which appears to be independent of common CAD risk factors, carries a worse prognosis ,64.At present there are no solid, prospective data on which to make definitive recommendations for treatment of concomitant conditions in patients with IPF.Acute exacerbations of IPF (AE-IPF) are, by definition, idiopathic. Therefore, known causes of acute deterioration, such as pulmonary embolism, congestive heart failure, pneumothorax and infection need to be thoroughly searched for and excluded. Based on data derived from recent randomized clinical trials, the yearly incidence of AE-IPF is between 10 and 15% of all patients ,65,66. TIPF is often initially misdiagnosed - at least until physiological and imaging data suggest the presence of an interstitial lung disease - leading to delay in accessing appropriate care. This point is not trivial in a disease with a median survival of 3 years after diagnosis. In a prospective cohort study of 129 IPF patients Lamas and coworkers showed that delayed access to a tertiary care center  is associated with a higher risk of death independent of disease severity . In turnCO < 39% predicted and/or evidence of a FVC decline > 10% over 6 months [Lung transplant (LTx) represents the only treatment of proven benefit in IPF. In fact, in IPF patients lung transplant has been shown to reduce the risk of death by 75% as compared with patients who remain on the waiting list . Since t6 months , but the6 months .Lack of energy and fatigue are common and disabling problems for patients with IPF. Pulmonary rehabilitation (PR) may alleviate symptoms and improve functional status by stabilizing and/or reversing the extra-pulmonary features of the disease ,79. TypiAlthough the key goal of treatment in IPF is to prevent disease progression, palliating symptoms such as dyspnea, chronic cough, depression and anxiety is equally important . In addiIn advanced disease dyspnea can be extremely distressing, thus impairing physical activity and quality of life. In selected cases of particularly severe dyspnea morphine could be considered. In a small case series Allen and colleagues reported that low dose diamorphine reduces dyspnea, anxiety and cough without significant decrease in oxygen saturation . FurtherTherapeutic challenges in IPF are summarized in Table inconsistent with UIP and histopathology showing UIP). Multidisciplinary specialist clinics and coordinated services can be only found in dedicated centers with high-volume ILD programs. Referral to centers with expertise may also allow participation in a clinical trial.The diagnosis and management of IPF represent a difficult task even for the most experienced clinicians. The disease should always be suspected in patients aged ≥50 years current or ex-smokers complaining of dry cough and dyspnea, particularly in the presence of compatible HRCT features . HoweverManagement of IPF is similarly challenging. The only care options that are endorsed by current guidelines are lung transplantation and enrolment in a clinical trial . In the The goals of any disease management strategy include improving quality of life, helping patients to be more functional, reducing disease-related morbidity and complications or helping people to live longer. Given the absence of a proven, effective therapy a large degree of therapeutic nihilism has always surrounded the management of IPF. However, a more recent trend focusing on improving symptoms and quality of life in IPF patients has been gaining ground, and it is now widely accepted that palliative care should be a treatment priority and an integral and routine component of patients care .Much work remains to be done in order to detect IPF in preclinical and early stages; nevertheless, the diagnostic accuracy has significantly increased by means of multidisciplinary discussion between clinicians, radiologists and pathologists with expertise in ILD. Similarly, and perhaps more importantly, recent clinical trials have shown promise in identifying treatment options. It is hoped that the results from ongoing and new clinical trials will soon determine an effective treatment for patients with this devastating disease.co: Diffusing capacity of carbon monoxide; ET: Endothelin; FPF: Familial pulmonary fibrosis; FVC: Forced vital capacity; GERD: Gastroesophageal reflux disease; HP: Hypersensitivity pneumonitis; HRCT: High-resolution computed tomography; ILDs: Interstitial lung diseases; IPF: Idiopathic pulmonary fibrosis; LAS: Lung Allocation Score; LTx: Lung transplant; NAC: N-acetylcysteine; NSIP: Nonspecific interstitial pneumonitis; PAH: Pulmonary arterial hypertension; PH: Pulmonary hypertension; PR: Pulmonary rehabilitation; 6MWD: 6-minute-walk distance; UIP: Usual interstitial pneumonia; VATS: Video-assisted thoracoscopy.ACE: Angiotensin-converting enzyme; AE: Acute exacerbations; ANA: Antinuclear antibodies; BAL: Bronchoalveolar lavage; CAD: Coronary artery disease; COPD: Chronic obstructive pulmonary disease; CTD: Connective tissue disease; DLLR reports receiving consulting fees from BoehringerIngelheim, Intermune, Celgene, Sanofi, Anthera and Gilead; lecture fees from Intermune. PS, RT, EC and AS have no conflict of interest to declare.PS and LR conceived of the study and drafted the manuscript. RT, EC and AS participated in the design of the review and helped to draft the manuscript. All authors read and approved the final manuscript.


Although CMR initially focused on the in vivo identification of myocardial infarction, recent work highlighted the advantage of CMR to provide more detailed in vivo tissue characterisation to help establish a differential diagnosis of the underlying aetiology.3Cardiovascular magnetic resonance (CMR) is well-established and increasingly used in clinical practice for the diagnosis of myocardial diseases.CMR have been performed routinely in some hospitals in the USA and other developed countries. However, there are few hospitals where CMR is performed for clinical practice in China. In our hospital, 285  patients with heart disease who underwent the comprehensive CMR  were admitted in cardiovascular department from January 2011 to April 2013. All of these patients were diagnosed with CMR . Interestingly, the most common disease was not myocardial infarction  but hypertrophic cardiomyopathy  in these patients. Other myocardial diseases included dilated cardiomyopathy , Myocarditis , hypertension , amyloidosis , restricted cardiomyopathy , arrhythmogenic right ventricular cardiomyopathy , hyperthyroid heart disease , left ventricle non-compaction , fabry disease  and other uncatelogued cardiomyopathy .3 the implementation is mainly limited by its long acquisition time and high costs in China. Further work needs to be done to establish CMR as an essential diagnostic tool for cardiologists. Although CMR can offer a unique non-invasive tool which should be integral part of the clinical work-up of a cardiomyopathy,


Indirect plant defenses are those facilitating the action of carnivores in ridding plants of their herbivorous consumers, as opposed to directly poisoning or repelling them. Of the numerous and diverse indirect defensive strategies employed by plants, inducible volatile production has garnered the most fascination among plant-insect ecologists. These volatile chemicals are emitted in response to feeding by herbivorous arthropods and serve to guide predators and parasitic wasps to their prey. Implicit in virtually all discussions of plant volatile-carnivore interactions is the premise that plants “call for help” to bodyguards that serve to boost plant fitness by limiting herbivore damage. This, by necessity, assumes a three-trophic level food chain where carnivores benefit plants, a theoretical framework that is conceptually tractable and convenient, but poorly depicts the complexity of food-web dynamics occurring in real communities. Recent work suggests that hyperparasitoids, top consumers acting from the fourth trophic level, exploit the same plant volatile cues used by third trophic level carnivores. Further, hyperparasitoids shift their foraging preferences, specifically cueing in to the odor profile of a plant being damaged by a parasitized herbivore that contains their host compared with damage from an unparasitized herbivore. If this outcome is broadly representative of plant-insect food webs at large, it suggests that damage-induced volatiles may not always be beneficial to plants with major implications for the evolution of anti-herbivore defense and manipulating plant traits to improve biological control in agricultural crops. Predation and herbivory are the two most commonly studied ecological interactions, in large part because of the sheer abundance and diversity of prey- and plant-feeding animals in nature. Until somewhat recently, however, the functional roles of predators and herbivores were considered to be largely independent of one another. This meant that those investigating the effects of predators on their herbivorous prey could do so with little to no consideration of plants, which were merely viewed as the substrate upon which predator-prey dynamics played out but not actively involved in the process. The past three decades of ecological research into terrestrial plant-animal communities has dramatically transformed this perspective. It is now widely accepted that plant-herbivore-predator, or tri-trophic, interactions represent a fully integrated and functionally interdependent unit in which plants facilitate carnivores Although plant traits modify carnivore function via numerous mechanistic routes , the lion's share of theoretical and empirical attention has gone toward volatile plant chemicals emitted in response to herbivore feeding damage that attract the enemies of those herbivores, otherwise known as the “call for help” Tri-trophic interactions mediated by plant volatiles are especially well documented for herbivorous insects, mostly caterpillars, because of their small size and thus intimate association with host-plants, which serve as food and housing . ConsequCommunity structure, population control, and competition” The relatively simple picture painted above  is the working conceptual model envisioned by virtually all researchers in this field. Is this mechanistic flow chart overly simplistic? Perhaps. The very nature of the term “tri-trophic interactions” is explicitly based on a three-trophic level system where plants, herbivores, and carnivores exist as three discrete groups and carnivores trigger a top-down trophic cascade . Community ecologists have been debating this vision of trophic dynamics for decades, beginning with Hairston, Smith, and Slobodkin's controversial paper “Although it is now recognized that plant volatiles are multi-functional, mediating interactions with pollinators, seed dispersers, neighboring plants, etc. PLOS BiologyBrassica oleracea, whose leaves are chewed by larvae of the cabbage white butterfly, Pieris rapae. From the third trophic level, two parasitic wasps, Cotesia rubecula and C. glomerata, lay eggs inside of and eventually kill the herbivorous P. rapae. And from the fourth trophic level, another wasp, Lysibia nana, parasitizes and kills the two Cotesia species (but not P. rapae). Wasps that parasitize other parasitic wasps are termed hyperparasitoids, a remarkably diverse and common group but whose ecology and behavior are poorly documented B. oleracea volatile profiles, and multi-year field experiments and natural population surveys, Poelman et al. offer strong evidence that hyperparasitoids exploit herbivore-induced plant volatiles in seeking out primary parasitoids as hosts. This finding alone would be a substantial contribution to the existing body of knowledge regarding induced defenses and carnivore attraction in a community context. Two novel insights, however, set this work apart.A new study by Poelman and colleagues published in this issue of E)-DMNT, for example, was 5.6, 7.5, and 15.2, respectively, from the undamaged control, plants damaged by unparasitized caterpillars, and plants damaged by C. glomerata-parasitized caterpillars. This means that plant biochemical responses quantitatively differ depending on the parasitism status of the herbivore, in this case the magnitude of (E)-DMNT induction was nearly five times greater from plants chewed by parasitized caterpillars. Although induced volatiles are known to differ across herbivore species, e.g., C. glomerata-parasitized  caterpillars. The authors speculate this putatively adaptive behavior is a consequence of hyperparasitoids realizing higher fitness on the gregarious C. glomerata compared with the solitary C. rubecula. Overall, these data beg the question—which trophic level is ultimately in the driver's seat? The herbivore or the parasitoid? While the acronym HIPV is often used as shorthand for “herbivore-induced plant volatile,” PIPV, for “parasitoid-induced plant volatile,” may soon enter the vocabulary of plant-insect ecologists!First, the authors report an astonishing level of specificity in plant and hyperparasitoid responses to damage by parasitized versus unparasitized caterpillars. Emission of the plant-derived terpenoid (A second key feature of the Poelman et al. study is its isolation and identification of mediating mechanisms. Parasitic wasps elicit many developmental changes in their caterpillar host, any number of which could be responsible for the above-described specificity patterns. For instance, parasitized herbivores consume less leaf tissue, a behavioral shift that would be expected to impact volatile production given that plant responses to herbivory tend to correlate with damage level. The authors employed an elegant technique whereby caterpillar oral secretions were exogenously applied to a standardized wound on the leaf surface to control for variable tissue damage. Remarkably, hyperparasitoid preference for volatiles of plants attacked by parasitized caterpillars was entirely mediated by salivary chemistry and the effect could be recreated by simply applying saliva from parasitized caterpillars to a leaf wound. Prior work in this Brassica fitness was not evaluated and, in fact, it is questionable whether the structure of this food web even allows for a top-down cascade of hyperparasitoids on plants. The wasp, L. nana, attacks primary parasitoids after they have already killed the herbivore P. rapae and emerged from their host's cadaver to spin cocoons and pupate. Thus, hyperparasitism in this system does not necessarily prevent primary parasitoids from protecting plants against caterpillar herbivory, at least on an individual plant basis. That being said, hyperparasitism could reduce wasp abundance at the population-level, making selection pressure on plant chemistry more diffuse and challenging to empirically track. As a whole, studies across plant-insect communities need to begin documenting the plant fitness consequences of variable volatile production and link this relationship with carnivore function, as pleaded for in recent reviews, e.g., Poelman et al.'s work clearly implies selection on fourth trophic level hyperparasitoids to detect subtle shifts in plant volatile constituents and take advantage of this information in host-finding behavior. What is far less clear is whether hyperparasitoids exert reciprocal selection pressure on plants. In the noted study, Anacharis zealandica, to turnip plots baited with methyl salicylate, the most commonly deployed plant volatile used in biocontrol. Because lacewings are voracious aphid predators, attraction of their parasitoid could indirectly aggravate pest outbreaks from the fourth trophic level. This scenario remains highly speculative, however, until we gain a better understanding of the potential for positive effects of hyperparasitoids and other top consumers on herbivores, which at present is limited Beyond the basic evolutionary repercussions of this work, the data also weave together a cautionary tale for manipulating agricultural crop traits to enhance the impact of natural enemies in biological pest control. Increasingly, plant volatiles are eyed as novel tools for augmenting predators and parasitic wasps, but, again, this application is entirely based on the three-trophic level concept


Effects of early life psychosocial adversity have received a great deal of attention, such as maternal separation in experimental animal models and abuse/neglect in young humans. More recently, long-term effects of the physical stress of repetitive procedural pain have begun to be addressed in infants hospitalized in neonatal intensive care. Preterm infants are more sensitive to pain and stress, which cannot be distinguished in neonates. The focus of this review is clinical studies of long-term effects of repeated procedural pain-related stress in the neonatal intensive care unit (NICU) in relation to brain development, neurodevelopment, programming of stress systems, and later pain sensitivity in infants born very preterm . Neonatal pain exposure has been quantified as the number of invasive and/or skin-breaking procedures during hospitalization in the NICU. Emerging studies provide convincing clinical evidence for an adverse impact of neonatal pain/stress in infants at a time of physiological immaturity, rapidly developing brain microstructure and networks, as well as programming of the hypothalamic-pituitary-adrenal axis. Currently it appears that early pain/stress may influence the developing brain and thereby neurodevelopment and stress-sensitive behaviors, particularly in the most immature neonates. However, there is no evidence for greater prevalence of pain syndromes compared to children and adults born healthy at full term. In addressing associations between pain/stress and outcomes, careful consideration of confounding clinical factors related to prematurity is essential. The need for pain management for humanitarian care is widely advocated. Non-pharmacological interventions to help parents reduce their infant’s stress may be brain-protective. The importance of pain in hospitalized newborns was first recognized in the 1980s. Prior to this time it was assumed that infants could not perceive pain early in life and that risks of pharmacological agents outweighed potential benefits. There were a series of seminal studies that began to define the field of infant pain. Concurrently, concerns about the developmental needs of very preterm neonates were raised.Evidence will be presented for long-term associations between repeated pain in the NICU in infants born very preterm (born ≤32 weeks’ gestation) and altered brain development, neurodevelopment, programming of stress systems, and later pain perception in infants born preterm. Given the extensive animal literature that has established mechanistic foundations for the impact of early environmental stress on the developing organism, together with the accumulating clinical evidence, it appears possible that exposure to prolonged and repetitive pain-related stress in infants born very preterm may potentially have long-term effects contributing to altered neurobehavioral development in vulnerable infants.During weeks to months in the NICU, very preterm infants are exposed to a high number of life-saving skin-breaking procedures and interventions, as well as routine handling that elicit behavioral, physiological, and hormonal responses. The immature peripheral and central nervous system of the very preterm infant responds differently to pain.Repeated pain/stress exposure in very preterm infants takes place at a time of rapid brain development and programming of the hypothalamic-pituitary-adrenal (HPA) axis. Synaptic connections are being formed, activity-dependent selective cell death (apoptosis) shapes the developing brain, and integrated cortical networks are becoming established.In the late second and third trimesters of fetal life, the period when the very preterm neonate born at 24–32 weeks’ gestation is in the NICU, the developing brain undergoes major changes in cytoarchitecture and development of functional networks. During this lengthy period of hospitalization of neonates born extremely preterm (≤28 weeks’ gestation) brain development includes establishment and differentiation of subplate neurons, alignment, orientation and layering of cortical neurons, elaboration of dendrites and axons, formation of synapses, selective pruning of neuronal processes and synapses, and proliferation and differentiation of glial cells.33Procedural pain/stress in very preterm infants is associated with abnormal brain development in the NICU, above and beyond other clinical risk factors associated with prematurity.Diffusely abnormal microstructure and metabolismSeveral mechanisms or interactions of different systems may potentially link neonatal pain-related stress exposure to altered neurodevelopment in this vulnerable population. The developing brain may be directly influenced via hemodynamic changes at a time of very immature autoregulation of cerebral blood flow, or indirectly through altered development of sleep/wake state architecture and programming of stress systems. Importantly, immature neurons are more sensitive to neurotoxic environmental influences.Recently Grunau, Miller, and colleagues specifically addressed in preterm infants whether neonatal procedural pain/stress impacts the developing brain. In a longitudinal study, infants born very preterm at 24–32 weeks’ gestation underwent advanced MRI brain imaging early in their NICU stay and again at term-equivalent age.The potential for procedural stress in the NICU to affect the brain adversely was demonstrated long ago, in a study reporting that endotracheal suctioning altered neonatal cerebral blood flow.52Stress of handling and procedures in the NICU is associated with changes in brain structure and function.At school-age, there appears to be only one study that has examined brain reactivity to painful stimuli in children born preterm. On functional magnetic resonance imaging (fMRI) at age 9–14 years, children born preterm displayed greater activation in the somatosensory cortex and other brain regions, compared to children born full-term with or without early hospitalization.Importantly, programs designed to recognize infant stress cues and provide supportive care, compared to standard practice, are associated with improved brain maturation. The Neonatal Individualized Developmental Care and Assessment Program (NIDCAP), compared to standard care of preterm neonates, led to more mature coherence between frontal and other brain regions on EEG and better neurobehavioral function.In a longitudinal cohort of preterm infants followed from birth to school-age, Grunau, Ribary, and colleagues examined whether neonatal pain is associated with functional brain activity later in childhood. They found that greater cumulative neonatal pain-related stress was associated with altered spontaneous oscillatory brain activity  at age 7 years.While there is now initial evidence for both direct and indirect relationships between repeated prolonged exposure to neonatal procedural pain and the developing brain, a great deal more research is needed to reveal the mechanisms and relationships with other risk factors of prematurity.While cerebral palsy has decreased among preterm infants in recent years,31Early pain-related stress may affect specific developmental domains via different systems. As described above, pain appears to affect cognition and motor function through changes to brain microstructure and function. In contrast, internalizing behaviors that include depressive, anxiety, and somatic symptoms—all stress-sensitive—may be more related to altered programming of the hypothalamic-pituitary-adrenocortical (HPA) axis. This distinction is somewhat arbitrary, however, given that cortisol levels are also involved in brain function. At 18 months’ CA, we found that cortisol levels were altered across the first two years of life in extremely preterm infants.69Relationships between physiological and behavioral reactivity to external stimulation such as touch or pain, the contribution of concurrent clinical events in the NICU such as hypotension, infection, and inflammation, and how these may interact to affect mechanisms underlying motor, cognitive, and complex behavioral development will require relevant animal models integrated with clinical research.Sleep architecture and sleep–wake states start to develop during the third trimester of fetal life. Sleep has an important role in brain development, and disturbances in sleep–wake patterns affect the developing central nervous system.The adrenal cortex releases the primary stress hormone cortisol (corticosterone in rodents) that contributes to adaptation under different levels of stress and to changes in brain function. A major literature extending for more than 50 years has established long-term effects of early social adversity, extending to the transformative contributions of Meaney and colleagues on the mechanisms of biological encoding of maternal behavior  at school-age in children born very preterm. The NFκBIA gene encodes IκBα, a critical negative regulator of the transcription factor NFκB.NFκBIA rs2233409 minor allele (CT or TT), greater neonatal pain-related stress (number of skin-breaking procedures from birth to term), independent of medical confounders, was associated with lower hair cortisol at age 7 years. Moreover, the minor allele of NFκBIA rs2233409 was associated with higher secretion of inflammatory cytokines, suggesting that neonatal pain/stress may act as a proinflammatory stimulus that induces long-term immune cell activation. These findings are the first evidence that a long-term association between early pain-related stress and cortisol may be mediated by a genetic variant that regulates the activity of NFκB, suggesting possible involvement of stress/inflammatory mechanisms in HPA programming, at least in boys born very preterm.There are complex bidirectional influences between the central nervous system, the HPA axis, and the immune system.Early studies used a parent questionnaire to measure pain sensitivity in preterm children. Parents reported lower pain sensitivity to everyday bumps, scrapes, and falls in micropremies born at or below 800 g compared to control children born full-term.Studies that have directly compared behavioral and physiological pain responses in former preterm compared to full-term infants long after NICU discharge revealed that age at testing is important. At 4 months’ CA, i.e. age adjusted for prematurity, infants born at or below 800 g (i.e. micropremies born below 26 weeks’ gestation) showed no differences in behavioral or cardiac responses to blood collection by finger lance, a site that had not been previously used and thus had no peripheral pain stimulation in the past.Later in childhood, there have been a number of experimental studies of pain threshold in children born preterm, revealing complex effects. Adolescents born preterm had more tender points and lower pain threshold compared to their term-born peers.In some other studies of long-term changes in pain sensitivity following early surgery, both preterm and full-term children have been included in samples. Pain threshold at school-age depended on type of surgery and whether threshold was tested in the region of surgery. For example, sensitivity among children who had chest surgery in infancy showed reduced sensitivity to touch, cold, and heat in the region of the surgery.98Pain threshold may be altered in children long after early surgery, especially in regions of prior tissue damage, which is important if another surgery is needed later. However, the direction and magnitude appear to depend on many factors that are unclear at this point, perhaps at least in part due to inclusion of children born preterm and full-term. It is unknown whether physiological immaturity at the time of surgery may contribute to the long-term impact on later touch or pain threshold.Although repeated procedural pain early in life in infants born preterm is associated with altered sensory thresholds when tested in experimental conditions, studies of self-report, however, suggest no differences in “everyday” pain or pain syndromes compared to full-term controls in adolescents or young adults born preterm.In a longitudinal randomized trial where mothers were taught how to reduce stress in their low-birth-weight infants, IQ scores at 9 years of age were >10 points higher in the intervention group.Potentially protective factors in the face of early biological adversity are recognized as a crucial focus to explain the wide variability of neurodevelopmental outcomes in very preterm infants.There is now convincing evidence that repeated neonatal procedural pain/stress in very preterm infants in the NICU may have the potential to adjust set points in biological circuits and alter brain microstructure and function, stress systems, neurodevelopment, and stress-sensitive behaviors. This suggests potential mechanisms that may contribute to the etiology of neurodevelopmental and behavioral problems in children born very preterm. Genetic variation contributing to diverse effects has just begun to be examined,Addressing whether specific approaches to pain management in the NICU may improve the developing brain and promote better long-term outcomes is urgently needed. While morphine does not appear to affect developmental outcomes adversely, pre-emptive continuous morphine infusion for pain management has yielded little if any benefit for prevention of morbidities and is no longer recommended. The burgeoning field of pharmacogenomics in future holds promise for individualizing pharmacologic pain management but has not yet been addressed with preterm infants. Currently, sucrose is widely used for routine minor procedural pain; however, there is a dearth of research into whether there are long-term positive or negative effects of repeated sucrose exposure in tiny babies. Supportive “environmental care” and parent involvement show promise for reducing stress in preterm neonates, thereby improving brain structure and activity. The extent to which non-pharmacologic pain management may prevent long-term effects of neonatal pain remains unknown.


Although several psychological and pharmacological treatment options are available for anxiety disorders, not all patients respond well to each option. Furthermore, given the relatively long duration of adequate treatment trials, finding a good treatment fit can take many months or longer. Thus, both clinicians and patients would benefit from the identification of objective pre-treatment measures that predict which patients will best respond to a given treatment. Recent studies have begun to use biological measures to help predict symptomatic change after treatment in anxiety disorders. In this review, we summarize studies that have used structural and functional neuroimaging measures to predict treatment response in obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), and social anxiety disorder (SAD). We note the limitations of the current studies and offer suggestions for future research. Although the literature is currently small, we conclude that pre-treatment neuroimaging measures do appear to predict treatment response in anxiety disorders, and future research will be needed to determine the relative predictive power of neuroimaging measures as compared to clinical and demographic measures. Anxiety disorders are highly prevalent  and are Given the variability in treatment response, it would be beneficial to determine whether measures obtained before treatment could help clinicians predict symptomatic change in response to a given treatment. Several clinical measures have been useful in this regard. For example, greater pre-treatment symptom severity has been associated with less favorable response to treatment in OCD , PTSD 11,12, socRecent studies have begun to use biological measures to predict symptomatic change after treatment in anxiety disorders e.g., -22). Suc. Suc22])The studies reviewed below implemented a variety of different imaging techniques and data analytic approaches. Early studies tended to use positron emission tomography (PET) and fluorodeoxyglucose (FDG) to measure rCMRglu while patients rested with eyes open. Although such resting state studies are relatively easy to administer because they require no explicit cognitive task, they are also limited by a lack of control over the mental processes in which subjects engage during the radiotracer uptake period. This can lead to greater variability in the data, perhaps increasing the risk of type II errors , as well as some difficulty interpreting the meaning of the rCMRglu findings. In general, PET-FDG imaging is also limited by relatively poor temporal resolution as rCMRglu data are typically averaged across long periods of time (~30-45 minutes).Many older PET-FDG studies also used region-of-interest (ROI) based analyses in which researchers draw boundaries around brain regions based on structural anatomy, “extract” functional imaging data from those drawn regions, and submit the extracted data to external statistical software. One disadvantage of this technique is that it involves averaging functional imaging data across very large brain structures, increasing the risk of type II error if only a small portion of that large brain structure is actually important in the prediction of treatment response. More recent neuroimaging studies employ voxelwise analyses that can assess the relationship between brain activation (or gray matter volume) and treatment response at every voxel  in the brain. However, in order to complete such analyses, one must “normalize” or morph the functional  brain images to a standard brain template, which introduces some amount of error. In addition, voxelwise analyses involve conducting thousands of analyses (one per voxel) across the brain, so correction for multiple comparisons must be applied.More recent studies have implemented functional magnetic resonance imaging (fMRI) to measure brain activation in response to cognitive and/or affective tasks. These types of studies afford more control over the cognitive state of the participant during scanning and, if well designed, allow for a more clear interpretation of the meaning of the imaging findings.The methodological considerations described above apply to most functional neuroimaging studies, not just those assessing the prediction of treatment response. With regard to the latter specifically, researchers have used two different data analytic approaches. The first approach involves comparing treatment responders to non-responders on a pre-treatment neuroimaging measure . For example, if treatment responders as a group were found to have lower pre-treatment amygdala activation than non-responders, then lower amygdala activation would be considered to be predictive of a better response to treatment. This type of between-group analysis typically involves analysis of variance (ANOVA) or analysis of covariance (ANCOVA), and requires (1) a very clear and well-accepted definition of treatment response and (2) sufficiently large numbers of participants per group (responders and non-responders). Because the relative size of these post-treatment groups cannot be completely planned a priori, these studies should begin with a very large number of participants.The second approach is to run a correlation (or a regression) between a continuous pre-treatment neuroimaging measure and a continuous measure of symptomatic improvement  improvement score). This type of analysis takes advantage of the inherent variability in both the imaging and treatment response data and is therefore likely to be more powerful than the between-group approach. Regression analyses also obviate the need to form responder and non-responder groups and permit the investigation of multiple pre-treatment imaging (and non-imaging) variables in the prediction of treatment response. For example, baseline symptom severity and comorbidity measures  can be entered into the regression along with pre-treatment imaging measures to predict symptom change scores. Several studies reviewed below included both types of approaches.change in neuroimaging measures with treatment as those studies address a different question.In the following text, we review studies that have assessed whether pre-treatment structural or functional neuroimaging measures can predict treatment response in OCD, PTSD, GAD, and SAD  and  and 67])In an fMRI study, Bryant and colleagues  presenteIn a voxel-based morphometry study of railway workers with PTSD, Nardo et al.  found thIn summary, pre-treatment neuroimaging measures of the amygdala and mPFC  predict response to BT/CBT in PTSD, although additional studies are needed to specify the direction of the findings. Interestingly, mPFC activation also appears to predict treatment response in GAD, as shown below.Although relatively few functional neuroimaging studies of GAD exist in the literature, there has been some support for increased activation in the amygdala and mPFC in GAD relative to comparison groups  activation is related to better response to venlafaxine.Functional neuroimaging studies of SAD have revealed exaggerated amygdala activation e.g. -79), whi, whi79])In a PET-FDG study, Evans and colleagues  found noDoehrmann and colleagues  used fMRIn summary, only two studies have examined neuroimaging predictors of treatment response in SAD, and those two studies differed quite dramatically in terms of imaging methods, sample sizes, treatment type, and findings. This variability and paucity of data prevent us from drawing general conclusions about the neuroimaging predictors of treatment response in SAD at the present time.In summary, although this literature is currently small, pre-treatment neuroimaging measures do appear to predict treatment response in anxiety disorders. Despite the variability in the findings within disorder categories, some consistent patterns have emerged. For example, in OCD, lower pre-treatment activity in the orbitofrontal cortex and greater activity in the caudate and posterior cingulate cortex appear to predict a more favorable response to treatment with SRIs or neurosurgery. In OCD, PTSD, and GAD, greater pre-treatment activity or gray matter density in the mPFC predicts better response to treatment . In PTSD and GAD, lower pre-treatment amygdala activation predicts a more favorable response to treatment (with CBT in PTSD and venlafaxine in GAD).Consistent with neurocircuitry models of OCD , caudatConfidence in these findings is tempered, however, by several limitations and caveats. First, as mentioned previously, the present literature is small and includes studies with relatively small numbers of subjects. Second, data analytic methods varied across studies and this alone could account for much of the variability in findings. Third, some studies did not appear to control for pre-treatment symptom severity, leaving open the question of whether neuroimaging measures are merely acting as proxies for baseline symptom severity in the prediction of treatment response. In order for neuroimaging measures to be practical in the prediction of treatment response, they would need to predict treatment response over and above the common clinical predictors, which are easier and less expensive to obtain. The findings of Doehrmann and colleagues  are espeFourth, because most studies did not use placebo control groups, it is possible that neuroimaging measures predict improvement in general rather than treatment response per se (as improvement could be due to expectancy effects). This may be less of a problem in studies that compared responders to non-responders if both groups had similar expectancies.Fifth, most of the studies reviewed above predicted response to one type of treatment. While this is a necessary first step, future studies will need to include more than one treatment type ,41 in orSixth, future studies will need to assess whether neuroimaging predictors of treatment response are specific to diagnostic categories. Furthermore, given the substantial comorbidity between anxiety disorders and depression, whether neuroimaging predictors of treatment response differ between these two diagnostic categories (and between comorbid and non-comorbid groups) will also need to be evaluated. A brief examination of the separate literatures reveals that some neuroimaging predictors of treatment response appear to be similar between anxiety disorders and depression  ,89, but Finally, arguably the most important big-picture question for future research to consider is whether the information gleaned from group studies (such as the ones described herein) can eventually be applied to clinical decision making in individual patients. Group studies could yield, for example, logistical regression equations or more complex classification schemes in which future individual patient data could be entered to predict a dichotomous treatment response outcome. This approach has been used in other areas of clinical research ,91, but ACC: Anterior cingulate cortex; BT: Behavioral therapy; CBT: Cognitive-behavioral therapy; dACC: Dorsal anterior cingulate cortex; fMRI: Functional magnetic resonance imaging; GAD: Generalized anxiety disorder; mPFC: Medial prefrontal cortex; OCD: Obsessive-compulsive disorder; PET: Positron emission tomography; PTSD: Posttraumatic stress disorder; rACC: Rostral anterior cingulate cortex; rCMRglu: Regional cerebral metabolic rate for glucose; rCBF: Regional cerebral blood flow; ROI: Region of interest; SPECT: Single photon emission computed tomography; SRI: Serotonin reuptake inhibitors.The authors declare that they have no competing interests.LMS read all studies reviewed herein and drafted the document. FCD, MBV, MKD, and SJD conducted literature searches and edited the document. All authors read and approved the final manuscript.


Lignin is a ubiquitous polymer present in cell walls of all vascular plants, where it rigidifies and strengthens the cell wall structure through covalent cross-linkages to cell wall polysaccharides. The presence of lignin makes the cell wall recalcitrant to conversion into fermentable sugars for bioenergy uses. Therefore, reducing lignin content and modifying its linkages have become major targets for bioenergy feedstock development through either biotechnology or traditional plant breeding. In addition, lignin synthesis has long been implicated as an important plant defense mechanism against pathogens, because lignin synthesis is often induced at the site of pathogen attack. This article explores the impact of lignin modifications on the susceptibility of a range of plant species to their associated pathogens, and the implications for development of feedstocks for the second-generation biofuels industry. Surprisingly, there are some instances where plants modified in lignin synthesis may display increased resistance to associated pathogens, which is explored in this article. Figure 1), in which lignin monomers are synthesized from the amino acid phenylalanine, then oxidatively polymerized into hydroxyphenol- (H-), guiacyl- (G-), or sinapyl- (S-) lignin. Lignin serves the critical function of reinforcing vascular elements for water transport under negative pressure; in severely lignin deficient plants, vascular collapse has been observed  into their sugar monomers, and converting the sugar into fuels. A third component of cell walls is the phenolic polymer lignin, which structurally fortifies the cell walls making them rigid and resistant to microbial degradation. Lignin content has been shown to negatively impact cellulosic bioenergy conversion via saccharification and fermentation to ethanol , which hobserved . Thus, tobserved . A critiZea mays) and sorghum (Sorghum bicolor), we have also included a review of the literature on effects of impairing steps in the monolignol pathway to pathogen responses in other plant species.There is a strong case for the involvement of lignin in plant defense. Lignin provides a physical barrier against initial ingress , and in Panicum virgatum), napiergrass (Pennisetum purpureum), sugarcane/energycane (complex hybrid Saccharum spp.), and miscanthus (Miscanthus × giganteus). Pathogens may pose a greater threat to perennial grasses as compared to annual row crops such as maize and sorghum, because production relies on establishment and harvest across multiple years before replanting, and the continual presence of the plants in the field provides refuge for the pathogens. Another factor that could impact plant–pathogen interactions is the level of genetic diversity within the field setting. Switchgrass varieties are maintained as an outcrossing population , antisense suppression of this gene led to increased susceptibility to the fungal pathogen Cercospora nicotianae, the causal agent of frogeye disease  was unchanged  signaling  is the first committed step in monolignol biosynthesis and the phenylpropanoid pathway. Altering the expression of this central gene has been shown to impact plant–pathogen interactions in model systems. In tobacco  and alfalfa (Medicago sativa), antisense/RNAi suppression of hydroxycinnamoyl CoA: shikimate transferase (HCT) one of the initial steps in monolignol biosynthesis, illustrates the potential of genetic/transgenic alterations to this pathway to constitutively activate defenses  HCT-RNAi; SID2 encodes an isochorismate synthase required for isochorismate-dependent SA synthesis. These results indicate that the stunted growth phenotype is due to elevated SA, occurring through an isochorismate-dependent pathway, rather than resulting from excess phenylalanine intermediates leading to the synthesis of SA in HCT-RNAi plants. Elevated levels of cold-water extractable pectin were correlated to elevated SA levels in transgenic alfalfa plants, which were RNAi-suppressed for six different genes (steps) in the monolignol pathway , the penultimate step in monolignol biosynthesis, was reported to increase resistance to pathogens or to have no effect on interaction with pathogens. In Arabidopsis, comt1 mutants show enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis, which is the causal agent of downy mildew  of the fungal pathogen Botrytis cinerea , RNAi suppression of the cinnamyl alcohol dehydrogenase (CAD) gene, the last step in monolignol biosynthesis, increased susceptibility to the pathogenic fungus F. oxysporum. A seedling assay showed the percent of infected seedlings was twofold higher in two CAD RNAi lines relative to WT ) relative to WT based on bacterial growth following inoculation  mutant, which encodes the ferulic acid 5-hydroxylase (F5H), last hydroxylase in monolignol synthesis, showed increased susceptibility to the fungal pathogen Sclerotinia sclerotiorum relative to WT in leaf assays (Triticum monococcum L.), five genes in monolignol biosynthesis were transiently silenced using particle bombardment of an RNAi vector containing PAL, caffeoyl-CoA O-methyltransferase (CCoAMT), F5H, COMT, or CAD genes (Blumeria graminis f. sp. tritici (host-specific) and Blumeria graminis f. sp. hordei (non-host). The silencing of all five genes individually and in pairs increased the susceptibility to both pathogens relative to the control bombarded with the empty RNAi vector, as determined by penetration efficiency of the fungus , it has been reported that no increased disease incidence were observed in antisense COMT or CAD lines relative to WT (brown midrib (bmr/bm) mutants of sorghum and maize (Zea mays), which have long been known to have reduced lignin content . Three Bmr loci have been cloned and characterized in sorghum. Bmr2, Bmr6, and Bmr12 all encode enzymes in monolignol biosynthesis: a 4-coumarate coenzyme A ligase (4CL), a CAD, and a COMT, respectively  were inoculated with Macrophomina phaseolina and lesion lengths were compared to corresponding WT lines , COMT (bm3/bmr12), and CAD (bm1/bmr6). The general trend from these studies indicate that maize and sorghum brown midrib mutants are not more susceptible to stalk rot pathogens, and in some cases show increased generalized resistance to specific pathogens.A study using inoculations of another fungal stock pathogen WT lines . Lesion  sorghum . Stalk sve to WT . Interesve to WT . Howeverbrown midrib mutants. Although the ability of these bmr plants to synthesize structural lignins is decreased and/or altered, there is no evidence bmr plants are impaired in their ability to synthesis “defense” lignin in response to pathogen attack, and the response might even be enhanced. Another explanation is that blocking a step in the lignin biosynthetic pathway would cause accumulation of lignin precursors and other phenolic compounds, because additional substrates would be available for their synthesis. Indeed it has been shown that some of these precursors inhibit the growth of pathogenic fungi or inhibit production of virulence factors (p-coumaric acid, and sinapic acid has been correlated with resistance to Fusarium spp. (bmr6 and bmr12 plants relative to WT (Arabidopsis and alfalfa (There are several explanations for the instances of increased generalized resistance observed in the  factors . For exaium spp. . We haveve to WT . Alterna alfalfa . A revie alfalfa , which dThese studies from a variety of plants indicate that reducing lignin content and altering its composition will not inevitably increase the susceptibility of bioenergy feedstocks to pathogens. There were not any clear trends that indicate that impairing a specific step in monolignol biosynthesis would affect plant susceptibility. In fact, studies from sorghum and maize indicate that impairing CAD or COMT activity in these lignin-modified plants showed more resistance to specific fungal pathogens, albeit these plants are not as resistant to the pathogen as resistant plant germplasm used in breeding efforts. In bioenergy feedstock species, modifications to monolignol biosynthesis will need to be evaluated on a case by case basis to determine the impact of pathogen susceptibility.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.


Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury. Neutrophils are armed with a broad repertoire of tools for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidant intermediates (ROIs). In addition to oxidant-dependent host defense, neutrophils harbor proteases, antimicrobial peptides, lactoferrin, and other antimicrobial constituents that damage and kill microbes. Activation of NADPH oxidase in neutrophils is linked to activation of intracellular granular proteases and to generation of neutrophil extracellular traps (NETs). NETs are composed of an extracellular network of chromatin bound to granular and specific cytoplasmic proteins that can target extracellular pathogens.in vitro by conditions that lead to robust NADPH oxidase activation , and is triggered in vivo during states of emergency, such as infection and conditions mimicking sepsis, such as transfusion-associated acute lung injury , damage-associated molecular patterns DAMPs; , and cytin vivo labeling study showed that the average estimated circulating human neutrophil lifespan was considerably longer  cytokine family], and is regulated by specific caspases and members of the Bcl-2 family of proteins . FactorsNETosis and necrosis of neutrophils are induced by different stimuli and have morphologically distinct features. Following toxin-induced necrosis, nuclear lobes in neutrophils lose their hypersegmented structure, but the nuclear envelope and granules remain intact . NuclearHow neutrophils die likely affects their clearance and cross-signaling to monocytes/macrophages. At sites of inflammation, neutrophils undergo spontaneous apoptosis . In addiNeutrophil extracellular trap generation was first described by NETosis can be triggered by exposure of neutrophils to PMA, IL-8, LPS, interferon-gamma (IFN-γ ), bacteria and fungi and their products, and complement-mediated opsonization . ActivatCandida  and p22phox. Upon activation, the cytoplasmic subunits, p47phox, p67phox, and p40phox and rac translocate to the cytochrome. NADPH is oxidized to NADP+, and electrons are transported down a reducing potential gradient that terminates when oxygen accepts an electron and is converted to superoxide anion. Neutrophil NADPH oxidase activation occurs in response to microbes and to stimuli that can mimic infectious threat, such as formylated peptides, opsonized particles, integrin-dependent adhesion , an inherited disorder of NADPH oxidase, is characterized by recurrent life-threatening bacterial and fungal infections. Patients with only residual NADPH oxidase activity in neutrophils have a better prognosis than those with completely absent oxidase function . CGD is in vitro . Therefore, deficiencies in these pathways can provide some insight into the role of NET constituents in host defense. The neutrophil serine proteases, NE, cathepsin-G (CG), and proteinase 3 (PR3), are activated by lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I DPPI; . Papillobscesses . MPO-def. For example, in patients with sepsis, the degree of NETosis may correlate with a better outcome due to enhanced bacterial clearance or a worse outcome reflecting inflammation-induced organ injury. Assays of NETotic markers  from cell-free fluid  may provide a quantitative measure of NETosis from archived human samples that could be correlated with clinical outcome in a variety of diseases. This knowledge may pave the way to therapeutic approaches to target NETs, such as use of histone neutralizing antibody or DNase1.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.


Sleep-wake disturbances and concomitant cognitive dysfunction in Parkinson’s disease (PD) contribute significantly to morbidity in patients and their carers. Subjectively reported daytime sleep disturbance is observed in over half of all patients with PD and has been linked to executive cognitive dysfunction. The current study used daytime actigraphy, a novel objective measure of napping and related this to neuropsychological performance in a sample of PD patients and healthy, age and gender-matched controls. Furthermore this study aimed to identify patients with PD who may benefit from pharmacologic and behavioural intervention to improve these symptoms.Eighty-five PD patients and 21 healthy, age-matched controls completed 14 days of wrist actigraphy within two weeks of neuropsychological testing. Objective napping measures were derived from actigraphy using a standardised protocol and subjective daytime sleepiness was recorded by the previously validated Epworth Sleepiness Scale.Patients with PD had a 225% increase in the mean nap time per day (minutes) as recorded by actigraphy compared to age matched controls . Significantly, differences in napping duration between patients, as recorded by actigraphy were not distinguished by their ratings on the subjective measurement of excessive daytime sleepiness. Finally, those patients with excessive daytime napping showed greater cognitive deficits in the domains of attention, semantic verbal fluency and processing speed.This study confirms increased levels of napping in PD, a finding that is concordant with subjective reports. However, subjective self-report measures of excessive daytime sleepiness do not robustly identify excessive napping in PD. Fronto-subcortical cognitive dysfunction was observed in those patients who napped excessively. Furthermore, this study suggests that daytime actigraphy, a non-invasive and inexpensive objective measure of daytime sleep, can identify patients with PD who may benefit from pharmacologic and behavioural interventions to improve these symptoms. Sleep-wake disturbance is gaining increased attention in Parkinson’s disease (PD). Such problems are observed in over two thirds of patients  manifestIn addition to daytime sleep disturbance, fatigue is frequently observed within PD cohorts. It is important to distinguish fatigue, an overwhelming lack of energy  from excThe development of daytime somnolence in PD has been associated with increasing age, disease duration, disease progression, postural instability, depression and the use of dopamine agonists ,19,20. HThe pattern of neuronal loss and neurotransmitter deficits giving rise to daytime sleep disturbance in PD are not well understood but dopaminergic and non-dopaminergic pathology across the brainstem, basal forebrain, hypothalamus and frontostriatal pathways have been suggested ,7,10,14.Previous studies in non-PD samples have shown that older individuals with EDS are more likely to nap during the day ,27. MoreStudies in non-PD cohorts have successfully utilised daytime actigraphy as a non-invasive measure of daytime napping ,36-38. FThis is the first study to compare the ESS, a widely used self-report questionnaire that rates the probability of napping, with an objective measure of napping. We hypothesised that the duration of daytime napping would be greater in PD patients as compared to controls and that excessive napping would be associated with impaired cognitive performance, specifically within domains mediated by fronto-subcortical circuitry. Furthermore we propose that as the ESS rates the probability of napping in several situations, patients with excessive daytime sleepiness as determined by the ESS, should also exhibit greater levels of napping as identified objectively by actigraphy Finally, we suggest that the objective measurement of daytime napping will more accurately identify those patients who may benefit from pharmacologic and behavioural interventions to improve these symptoms.Permission for the study was obtained from the University of Sydney Human Research Ethics Committee (HREC 02-2008/11105). All patients gave written informed consent.Eighty five patients and 21 age matched healthy controls were recruited from the Brain & Mind Research Institute (BMRI) PD Research Clinic, University of Sydney. All participants with a known or suspected diagnosis of obstructive sleep apnea were excluded, including any participant who had previously had CPAP prescribed or who had greater than mild OSA on a diagnostic sleep study . PatientAll neurological and neuropsychological assessments were conducted within one session to confirm study eligibility. Patients were assessed in their ‘on’ state and levodopa dose equivalents were calculated for dopaminergic medication . DiseaseNeuropsychological functioning was assessed within the PD cohort using standardised tests and appropriate normative data (with corrections for age and level of education). These variables were included in the healthy control group for descriptive purposes only. Language generativity was assessed with semantic verbal fluency via the Controlled Oral Word Associated Test  ,45. Set-The use of actigraphy to assess daytime sleep has been validated previously in healthy subjects in both the laboratory and community setting ,38 and tPatients were asked to complete the Epworth Sleepiness Scale (ESS) (score ≥ to 10 indicative of a high probability of daytime sleep) , within Statistical analysis was conducted on PASW Statistics Version 20 for Windows. Age was compared between the groups using a t-test. Gender was compared using a chi-square test. Subsequent variables violated assumptions of normality and non-parametric Mann-Whitney U test were used for these comparisons. All tests were two-tailed with an α value of 0.05. The three cognitive variables were compared between groups utilising a Bonferroni correction for multiple comparisons.As shown in Napping data shown in As mood disturbance has been linked previously to daytime somnolence , an anal2 = 0.194, p = 0.660). Similarly, global cognition (MMSE), mood disturbance (BDI-II), retention of learned verbal memory , and working memory (Digit Span backwards) were not different between the groups. As age was noted to be different between the two sub-groups of PD patients, age-adjusted normative z-scores were used. As shown in 2 = 0.294, p = 0.587).By contrast, differences in napping duration between patients with and without excessive daytime napping as recorded by actigraphy were not distinguished by their ratings on the ESS. This study is the first to use actigraphy, a previously validated objective measure of daytime sleep, to record the duration and correlates of excessive daytime napping in PD. Patients with PD, reported significantly greater number of nap bouts as well as time spent napping in the day, compared to healthy age matched controls. Patients with PD who exhibited excessive napping through the day had poorer performance on neuropsychological tests probing fronto-subcortical functions including set-shifting, semantic verbal fluency and processing speed. This result is in keeping with previous findings evaluating excessive daytime somnolence . HoweverThese results were not accounted for by age, mood disturbance, dementia, disease duration, disease stage or levodopa dose equivalent. As patients with obstructive apnea were excluded from the trial, this common cause of daytime sleepiness could not explain the excessive daytime napping seen in the PD group. Furthermore there was no evidence of sleep deprivation or poor sleep quality in patients compared to controls that may be an alternative cause of the increased daytime napping seen. Of note there was no difference in total sleep time, sleep efficiency or wake after sleep onset between patients with PD and controls. This finding is consistent with previous studies in this area although mixed results have been reported, which may reflect issues of sample size  and wrist actigraphy confirm the need for more objective measurement of daytime sleep-wake disturbance. Further, those patients with PD who nap excessively during the day have greater cognitive deficits in the domains of attention, semantic verbal fluency and processing speed. These results highlight a possible interrelationship between sleep and cognitive circuitry in PD that may represent common pathology. Further studies are now needed to evaluate the effect of prescribed napping, targeted at those with excessive daytime napping. Furthermore, given the potential for pharmacological and behavioural interventions to reduce excessive napping, trials are needed to investigate if these treatments can improve focal cognitive deficits in PD. This would have far reaching benefit to the quality of life of patients and their carers, in addition to reducing the burden of illness in the community.