Fecal contamination by human and animal pathogens, including viruses, bacteria, and protozoa, is a potential human health hazard, especially with regards to drinking water. Pathogen occurrence in groundwater varies considerably in space and time, which can be difficult to characterize as sampling typically requires hundreds of liters of water to be passed through a filter. Here we describe the design and deployment of an automated sampler suited for hydrogeologically and chemically dynamic groundwater systems. Our design focused on a compact form to facilitate transport and quick deployment to municipal and domestic water supplies. We deployed a sampler to characterize water quality from a household well tapping a shallow fractured dolomite aquifer in northeast Wisconsin. The sampler was deployed from January to April 2017, and monitored temperature, nitrate, chloride, specific conductance, and fluorescent dissolved organic matter on a minute time step; water was directed to sequential microbial filters during three recharge periods that ranged from 5 to 20 days. Results from the automated sampler demonstrate the dynamic nature of the household water quality, especially with regard to microbial targets, which were shown to vary 1 to 2 orders of magnitude during a single sampling event. We believe assessments of pathogen occurrence and concentration, and related assessments of drinking well vulnerability, would be improved by the time‐integrated characterization provided by this sampler. Article impact statement: Using the automated sampler described here, microbial concentrations in groundwater are shown to vary significantly over short time periods. Human and animal pathogens are disease‐causing microorganisms that can affect suitability of the water for certain use, and thus deteriorate groundwater resources and related water availability. Fecal contamination by human and animal pathogens, including viruses, bacteria, and protozoa, is a potential human health hazard, especially with regards to drinking water sources  deep and the casing extended to 8.8 m (29 feet) as determined by geophysical and borehole video logs . The well construction report for a replacement well  indicates that 6.7 m (22 feet) of surficial sediment overlie the dolomite at this site. The sampler was deployed January 18 through April 9, 2017.Bacteroides, ruminant Bacteroides, pepper mild mottle virus, and rotavirus group A. In the auto‐sampler work, we chose to target these frequently detected microbial markers as well as commonly reported total coliform concentrations.Concurrent with auto‐sampler deployment, a companion study was being conducted in the same region on the sources of fecal contamination in private wells. Four microbial targets were frequently being detected in the companion study: HF183 To provide context for automated sampler design decisions, a short description of a commonly employed manual sampling method for pathogens and indicators is provided, followed by a detailed description of the automated sampler workflow. See Appendix Microbial samples from groundwater are typically collected in two ways. Pumping wells are sampled by connecting a filter to a wellhead tap while the pump was running or piezometers are sampled by peristaltic or other pump with the tubing sterilized between samples. Viruses are concentrated in the field by dead‐end ultrafiltration, which has a pore size of approximately 30‐kDa  was met the system would be shut down by closing the main valve into the sampler. If glass‐wool filters were used, the automated sampler design described here could be modified to accommodate pH conditioning , and turbidity using a multi‐probe water‐quality sonde and a flow‐through chamber. Measurements were made every minute and stored on the sampler's datalogger. Water‐quality data collected were available real‐time using the two‐way telemetry of the sampler. The purpose of this monitoring was to characterize the dynamics and conditions of the household system during time periods when pathogen samples were taken. Because only periodic calibration was performed during the study, reported values are considered to approximate actual analyte concentrations. During initial testing, degassing of the water in the flow‐through chamber caused bubble artifacts in results from the water‐quality sensors (discussed below); orientating the sonde more vertically reduced, but did not eliminate, bubble formation. Water was continually flushed through the flow‐through chamber at a rate of approximately 0.6 L/min. This resulted in a constant relatively low‐flow rate discharge that required constant disposal using gravity drainage into a floor drain. Because the sampler is designed for installation in domestic households it included an emergency shutdown capability triggered by the occurrence of water in the bottom of the sampler. The sampler was constructed using a small refrigerator; therefore, filters could be refrigerated until they were removed to increase holding times.Hydrologic conditions used to determine when to activate microbial samplers were tracked using online resources. The regional groundwater system was monitored using real‐time data available from a USGS monitoring well .The local conditions in the household water supply were monitored on a minute time step using low‐flow rates routed through the water‐quality sonde flow‐through chamber. When a target recharge event was identified in the monitoring well hydrograph, the smartphone was used to open the valve to the first filter. The sampler program then monitored the flow rate until the user‐specified volume passed through the filter, at which point the valve to the first filter was shut and the valve to the second filter was opened. This continued until all filters were used or the user canceled the sampling event. Filters were collected every 1 to 3 days during the testing to minimize holding times and maintain continual coverage over the sampling period. During collection, the sampler was paused, filters with quick disconnect couplers were replaced, used filters placed in a plastic bag, sealed, and shipped to the analytical laboratory. Protective latex gloves were worn throughout filter collection to prevent contamination.Our initial discussion focuses on insights gained from the entire January to April 2017 sampling period; a March to April 2017 snowmelt event within this period is examined in more detail. All water quality data shown here are provided by Owens et al. .Data from the USGS monitoring well shows the period had multiple recharge events Figure . The samAlthough the water quality sonde data were only used to provide background characterization and identify potentially anomalous conditions in the household water system, results are included here for illustrative purposes. Water quality measured by the sampler's sonde show fluctuation operating on the order of days and weeks Figure . This isBacteroides was not found in any sample during the period. Total coliform, found in water from the well in all but one filter, can vary 1 to 2 orders of magnitude during sampling events . However, inclusion of a water quality sonde in the sampler provides a cost‐effective geochemical context for the water supply over time. Such insight is important for choosing microbial sampling periods, which helps ensure representative results while lowering the cost of microbial sampling.Most household water systems are under pressure, therefore artifacts of bubble formation during depressurization/degassing are expected to occur unless steps are taken to keep the flow‐through chamber pressurized.Inadvertent failure to restart the flow to the flow‐through chamber after sonde calibration is not flagged by the datalogger so gaps in the sonde time series can occur  to ensure a target system is adequately characterized.The results presented here demonstrate that microbe occurrence and concentration time series can be efficiently characterized in the field. These results represent those of shallow fractured aquifers with rapid travel times. In this setting, the sampler documents highly variable domestic household water quality over time. Characterization of groundwater quality using single snapshot sampling can be expected to leave longer‐term characterization of pathogen occurrence and magnitude highly uncertain. Therefore, deploying samplers as described here can be critical for understanding pathogen source and transport, as well as microbial dynamics, in aquifer settings.The authors do not have any conflicts of interest or financial disclosures to report.Appendix S1. Methods used for microbial sample processing and analysis.Click here for additional data file.


Although osteoma is a common benign tumor of the paranasal sinuses, its orbital extension is not common. Secondary orbital cellulitis has rarely been reported in association with sino‐orbital osteoma.A 30‐year‐old woman presented with left side proptosis, orbital pain and inflammation. Orbital CT scan showed a well‐defined giant osteoma in the superonasal part of the left orbit originating from the left ethmoidal sinus associated with opacity of the ipsilateral ethmoidal sinus and infiltration of orbital soft tissue. After treatment by systemic antibiotics, osteoma was resected with combined external and endoscopic surgery and the patient recovered uneventfully.Sino‐orbital osteoma may manifest primarily as orbital cellulitis and needs early surgical intervention. She also complained from headache, general weakness and fever. She had a history of congenital strabismus in the left eye but no history of head trauma.On general examinations, her temperature was 38.5°C. Her corrected visual acuity was 10/10 and 7/10 in the right and left eyes, respectively. She had a 5‐mm non axial proptosis and a temporal and downward displacement of the left‐sided globe. In addition to conjunctival injection and chemosis, the eyelids were edematous and inflamed in the left eye. Ocular movements of the left eye were limited in the upward and inward directions. Other ocular examinations including anterior and posterior segments were unremarkable. The afferent pupillary defect was negative Figure .Orbital CT scan showed a dense, heterogeneous, well‐defined and lobulated mass in the superonasal part of the left orbital extraconal space at the common border of the frontal bone and ethmoidal sinuses. As an anatomic variation, frontal sinuses were not pneumatized and left ethmoidal sinus was partially involved by the tumor. Tumor had displaced the globe to the inferotemporal space and exerted a pressure effect on the left medial rectus and optic nerve. Other findings were opacification of the left ethmoidal sinus and infiltration of the left orbital soft tissues Figure .The patient was admitted with the diagnosis of orbital cellulitis and underwent medical treatment with intravenous antibiotics including Ceftriaxone  and Vancomycin (Afachemi) 1 g every 12 hours each.After a 3‐day systemic treatment and partial resolution of the orbital inflammation, at first the patient underwent an endoscopic approach for tumor excision under guidance of a navigation system by a team consisted of otolaryngology and oculoplastic surgeons. After left ethmoidectomy, the ethmoidal part of the tumor was released from its connections in the sinus via the transnasal endoscopic approach. Because of the high density and large size of the tumor, the en‐block excision was only possible when the external approach via the modified Lynch skin incision was added to the endoscopic method. The excised tumor was a polypoid dense white mass measuring 3.5 × 2 × 1 cm. Histopathological examinations disclosed a dense lamellar mature bone tissue with numerous peripheral osteoblasts and interosseous fibrovascular tissues, consistent with a cancellous type osteoma (Figure There was no evidence of cerebrospinal fluid rhinorrhea post‐operatively. In addition to improvement in ocular movements, the proptosis and globe displacement were completely resolved after surgery but the congenital exotropia remained unchanged. Skin incision healed beautifully and no significant scar was seen Figure .The patient was also evaluated for Gardner syndrome by a gastroenterologist and her gastrointestinal endoscopic examinations were unremarkable. In follow up CT scan 2 years after the surgery, there was no evidence of tumor recurrence and the left orbital bony walls were intact excepting the lamina papyracea Figure . The pat3In this article we reported a rare case of orbital cellulitis secondary to a giant sino‐orbital osteoma and its successful management.Osteoma is a slow‐growing mesenchymal tumor which often involves periorbital sinuses in the craniofacial area.Most of the sinus osteomas are asymptomatic and can be an accidental finding of craniofacial imaging.Signs and symptoms of orbital extension of sinus osteoma can be divided into two categories. The first category is the result of pressure effect of the tumor including proptosis, orbital pain, diplopia, decrease in visual acuity, palpable mass and epiphora. The second category includes clinical findings secondary to sinusitis which may be the result of obstruction or damage to the involved sinus and presenting as orbital emphysema, orbital cellulitis and subperiosteal abscess formation adjacent to the involved sinus.Trauma, infection and developmental anomalies are suggested as the etiologic background of osteoma. In rare cases it may be seen in a genetic background such as Gardner syndrome that was rolled out in our case.For symptomatic cases of sino‐orbital osteoma specially giant osteomas, as reported in our patient, different surgical approaches are recommended from a pure external approach to a complete endoscopic technique.In cases with obstructed sinus openings and development of sinusitis, mucocele or cellulitis in the adjacent tissues, repair of the sinus drainage pathway and returning the sinus aeration, similar to what performed in our case, are mandatory.Although partial removal of the sino‐orbital osteomas was reported to be successful in many cases but tumor recurrence was reported in a few cases.In conclusion orbital cellulitis is a rare complication of sino‐orbital giant osteomas, which needs urgent intervention to not only treat the infection but also to remove the tumor and establish the sinus drainage.The study protocol was approved by the scientific and ethics committee of the Ophthalmic Research Center at Shahid Beheshti University of Medical Sciences and adhered to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from patient to use her medical data and face photographs.The authors have no financial interest in the subject of this article.Abbas Bagheri: Conceptualization; data curation; investigation; project administration; supervision; writing‐original draft; writing‐review and editing. Mohadeseh Feizi: Data curation; investigation; software; writing‐original draft. Reza Jafari: Data curation; investigation; writing‐original draft. Mozhgan Rezaei Kanavi: Data curation; investigation; methodology; supervision; writing‐review and editing. Nasim Raad: Conceptualization; data curation; methodology; validation; writing‐original draft.


The World Health Organization International Code of Marketing of Breast‐milk Substitutes prohibits claims and other marketing that may confuse caregivers about benefits of formula and other milk‐based drinks for infants and toddlers, but such marketing is common in the United States. This study assessed caregivers' provision of milk‐based products to their infants and toddlers and potential confusion about product benefits and appropriate use. Online survey of 1,645 U.S. caregivers of infants (6–11 months) and toddlers (12–36 months). Respondents identified infant formula and toddler milk products they served their child (ren) and provided relative agreement with common marketing claims. Logistic regression assessed relationships between agreement and serving these products, controlling for individual characteristics. Over one‐half of caregivers of infants (52%) agreed that infant formula can be better for babies' digestion and brain development than breastmilk, and 62% agreed it can provide nutrition not present in breastmilk. Most caregivers of toddlers (60%) agreed that toddler milks provide nutrition toddlers do not get from other foods. Some caregivers of infants (11%) reported serving toddler milk to their child most often. Agreement with marketing claims increased the odds of serving infant formula and/or toddler milks. For caregivers of toddlers, odds were higher for college‐educated and lower for non‐Hispanic White caregivers. Common marketing messages promoting infant formula and toddler milks may mislead caregivers about benefits and appropriateness of serving to young children. These findings support calls for public health policies and increased regulation of infant formula and toddler milks. Infant formula and toddler milk advertising may substantially affect caregivers' choice of milk for their child.Most caregivers believed marketing claims promoting unsubstantiated benefits of infant formula and toddler milks.Parents who believed these marketing claims were more likely to serve infant formulas or toddler milks to their child.Advertising that suggests infant formula and toddler milks provide developmental and other benefits for young children may neutralize public health messages and obscure health risks.Awareness of recommendations to offer toddlers whole milk and avoid sugary beverages did not prevent parents from providing toddler milks.Common infant formula and toddler milk marketing claims may mislead caregivers about product benefits and appropriateness for their child; public health campaigns to counter marketing claims and regulation of toddler milk labels are required.1The World Health Organization (WHO) enacted the International Code of Marketing of Breast‐milk Substitutes (the Code) in 1981 to address inappropriate and potentially harmful marketing of breastmilk substitutes (BMS). The Code aims “to contribute to the provision of safe and adequate nutrition for infants, by the protection and promotion of breast‐feeding, and by ensuring the proper use of breast‐milk substitutes, when these are necessary, on the basis of adequate information and through appropriate marketing and distribution” . Specific study objectives include measuring caregivers' provision of infant formula and/or toddler milks to their children; documenting the proportion who provided a product not intended for their child's age ; and assessing caregivers' agreement with common marketing claims and expert recommendations about milk‐based products for young children. We hypothesized that agreement with common infant formula and toddler milk marketing claims would be associated with caregivers' provision of these products to their young children.2N = 1,645) to obtain a diverse cross‐section of participants.We conducted a cross‐sectional online survey of U.S. parents and other primary caregivers of infants (6–11 months) and toddlers (12–36 months) to assess (a) the food and drink products caregivers served their child ; (b) caregivers' agreement with expert recommendations on how and what to feed their child; and (c) caregivers' agreement with marketing messages (including product claims) used to promote infant formula and toddler milks. We recruited a large nonprobability sample  to participate in the survey and sent them a link to the survey if they agreed (participation was voluntary). The survey screened for primary caregivers of at least one 6‐ to 36‐month‐old child who had decision‐making responsibility regarding what to feed their child. We established quotas to achieve equal numbers of respondents by child's age group , as well as by minimum numbers of Black and Asian caregivers  and higher and lower acculturation levels (Hispanic panel). Due to relatively low prevalence of some demographic groups  within the U.S. adult population, quota sampling was used to recruit a sufficient sample to allow for meaningful comparisons between all groups of interest . Participants with more than one eligible child selected the child with the most recent birthday and answered questions about that child. We divided participants into three groups according to the age of the child they selected: infants (6–11 months), young toddlers (12–23 months), and older toddlers (24–36 months).Participants reported the age and gender of child (ren) living in their household between 6 and 36 months of age. Children with a disease or condition that requires a special diet  were excluded  and similarity of brand names for different products , first we assessed the categories of milk‐based products that participants served their child. We then used a two‐step process to identify the specific infant formula or toddler milk product they served most often.all categories of milk drinks they had served their child in the past month, from four options: “Infant formulas ,” “Other formulas or powdered milks ,” “Regular milk ,” and “Non‐dairy milk .” The survey provided examples of infant formula and toddler milk brand names for clarity. Due to the lack of a commonly used term for “toddler milks,” the survey described them as “Other formulas or powdered milks.” The survey also included options for “Other” (with a fill‐in box) and “None of the above” (including if they only served breastmilk).Participants first selected product served most often, if participants selected “Infant formulas” and/or “Other formulas or powdered milks” in the category question, the next question showed a list of infant formula and toddler milk brand names and logos. Participants then selected all brands they had served their child in the past month. The list of brand names and logos included all infant formula and toddler milk brands that had spent $100,000 or more in total advertising in 2015, as identified in previous research  offered by each of the brands selected, and they chose the product they had served most often in the past month to their child. The list included all individual products offered by the brands in the previous question , but excluded products for specific dietary needs . We also identified participants who selected a product that was not intended for their child's age  as the one provided most often to their child.2.3.3strongly disagree to strongly agree. Specific recommendations and claims presented in the survey are included in the results.Participants also indicated their agreement with various statements and claims about infant formulas, toddler milks, and other drinks for either infants or toddlers (according to their child's age) using 7‐point Likert scales from disagree (1–3), neutral (4), or agree (5–7). Due to high covariance between the four infant formula marketing claims (α = 0.81), we averaged them to create one scale for agreement with infant formula claims.The survey included two statements each regarding expert recommendations for breastfeeding (shown to caregivers of infants [6–11 months]) or drinks for toddlers (shown to caregivers of toddlers [12–36 months]). Survey participants with an infant provided their agreement with four marketing claims commonly found on regular infant formula packages. Toddler caregivers saw one toddler milk claim (adapted from common claims on toddler milks) and a factual statement that toddler milks often contain added sweeteners . This validated tool provides a score that assesses language preference, ranging from 1 (only Spanish) to 5 (only English). In accord with SASH methodology, we classified Hispanic participants as less acculturated (score <3.0) or more acculturated , 3Of 2,581 original participants, 23% did not meet inclusion criteria. The final sample totalled 1,645, for an 82% completion rate. The sample was mostly female (80%), with approximately equal numbers by child age group , 22% of caregivers of infants (6–11 months) indicated that they had provided “other formulas”  in the past month. In addition, 50% of caregivers of toddlers indicated they had served their child infant formula. A smaller number of toddler caregivers (41%) indicated that they had served other formulas  in the past month. Thirty‐nine percent of all toddler caregivers served regular and/or non‐dairy milks in addition to infant formula and/or toddler milks products; whereas 29% served only regular and/or non‐dairy milk only.3.1When asked to select the specific product they served most often in the past month, not all caregivers indicated a product that was appropriate for their child's age. Among all caregivers of infants surveyed, 11% chose a toddler milk as the product they served most often. However, 53% had only selected “infant formula” when asked what categories of milk products they served, indicating that they may have believed the toddler milk product they served most often was an infant formula. Among toddler caregivers surveyed, 39% chose an infant formula as the product they served most often, but only 78% of these participants correctly categorized the product they had served as an infant formula.3.2M = 4.6, SD = 1.4).The majority of caregivers agreed with all expert recommendations and marketing claims Table . Althoug3.3Table strongly disagree] to 7 [strongly agree]). The odds of serving infant formula/toddler milk were four times higher if caregivers also served regular milk to their infant and 8.5 times higher if they also served non‐dairy milk. Neither agreement with expert recommendations, parent education, or race/ethnicity, nor infant age in months were associated with serving infant formula/toddler milk.In the model for caregivers of infants, when holding all other variables constant, the odds of serving infant formula/toddler milk increased by 45% with each point on the scale of agreement with infant formula marketing claims  participants were more likely to serve these products compared with non‐Hispanic White caregivers. Odds also decreased by 5% for every additional month of their child's age. In addition, odds were significantly lower if caregivers served their child regular milk (OR = 0.18). However, neither agreement that children should drink plain whole milk nor understanding that toddler milks contain added sweeteners was significantly related to caregivers' provision of infant formula/toddler milks to toddlers.4To our knowledge, this study is the first to demonstrate a relationship between agreement with infant formula and toddler milk marketing claims and whether caregivers served these products to their infant or toddler. Although the majority of participants agreed with expert recommendations to serve breastmilk to infants and plain whole milk to toddlers, it appears that they do not believe that those recommendations contradict common marketing claims about the benefits of infant formula over breastmilk and toddler milks over other food and drinks, as the majority of participants also agreed with marketing claims. Demographic characteristics were not related to serving infant formula/toddler milk to infants, but more educated caregivers, as well as non‐Hispanic Black, Asian, and more‐acculturated Hispanic caregivers, were more likely to serve them to their toddlers.These results also provide additional evidence that caregivers may be inappropriately serving toddler milks to infants. In this sample, 11% of infant caregivers reported serving a toddler milk product most often to their infant. This practice raises substantial concerns as toddler milks do not contain the required nutrition for infants . However, it is not clear why less‐acculturated Hispanic caregivers were not more likely to serve these products.In addition to their increased likelihood to serve infant formula/toddler milks to their child, toddler caregivers with a college degree in this sample were more likely to agree with the toddler milk marketing claim. This finding indicates that misperceptions about the meaning of marketing claims are not due to lower levels of education or literacy. In addition, the finding that non‐Hispanic Black, more‐acculturated Hispanic, and Asian caregivers were more likely to report serving infant formula/toddler milk to toddlers supports research in other domains suggesting that minority consumers may be more influenced by brand marketing than non‐Hispanic White consumers  for children up to 12 months. In contrast, toddler caregivers who served regular milk were less likely to serve infant formula/toddler milk, which indicates that caregivers may perceive toddler milk products to be appropriate substitutes for regular milk.4.1Although most caregivers in this survey agreed with expert recommendations about the superiority of breastfeeding over infant formula and the need to introduce young children to plain milk at approximately 1 year and avoid products with added sugar, they also agreed with common marketing claims that compare infant formula favourably to breastmilk and promise nutritional benefits from serving toddler milk. This finding suggests a need for health care providers and the public health community to specifically refute these marketing claims and clearly communicate that infant formula and toddler milk have no advantages over breastmilk and regular milk. The 2019 consensus statement by leading U.S. health organizations, including the American Academy of Pediatrics and the Academy of Nutrition Dietetics, specifically recommended against serving toddler milks, which represents an important first step. Similar guidance about infant formulas that promote unproven claims about benefits for common infant conditions, such as gas and fussiness, also appear to be necessary. Furthermore, public health campaigns and health care provider training should address and attempt to correct caregivers' misunderstandings about potentially misleading claims used in infant formula and toddler milk marketing.4.2better than breastmilk, suggesting that these claims are misleading or deceptive.These findings also demonstrate the need for more effective regulation of infant formula and toddler milk marketing in the United States. The FDA has the authority to regulate product labelling and could take action to address potentially misleading marketing claims and confusion about the difference between infant formula and toddler milk  and ascertain the exact products that participants served their child, and pretesting indicated participant understanding of category names used , this online survey did not allow for additional probes to assess participants' understandings. In addition, as with all self‐reported data, responses are subject to potential recall and/or desirability biases.Strengths of this study include the use of online survey panels, which offer geographic heterogeneity and the ability to assess differences by participant race/ethnicity, education, and income through sampling quotas. In addition, inclusion of actual brand and product names and images likely reduced misreporting of products served. Additional research is required to demonstrate that marketing claims caused caregivers to serve infant formula or toddler milk to their young child and that cross‐branding caused confusion about the appropriate products for their child's age. However, this study suggests that marketing of infant formula and toddler milk may substantially affect caregivers' decisions about the milk‐based products they serve their children.4.4Despite awareness of expert recommendations and public health efforts to promote breastfeeding for infants and plain whole milk for toddlers, the majority of caregivers agreed with common marketing claims that serving infant formula or toddler milks benefits their child. In addition, these messages may mislead caregivers to inappropriately provide them to young children. These findings support calls for increased regulation of marketing for infant formula and toddler milks. Public health organizations should provide additional guidance to caregivers about appropriate milk‐based products for infants and toddlers and highlight issues with potentially misleading marketing claims that imply these products are nutritionally superior and provide benefits over recommended milk‐based products for young children.The authors declare that hey have no conflicts of interest.MJR‐P and JLH were responsible for the conception and design of the study, and acquisition of data. JLP also participated in the analysis and interpretation of data, drafting, and revising the manuscript. All authors have approved the version for publication.Data S1 Supporting InformationClick here for additional data file.


Liposarcoma is a malignant adipose tissue tumor which mainly originates from the extremities and retroperitoneum. Primary pleural liposarcoma is very rare. Spindle cell lipoma is a rare benign adipose tissue tumor. A 66‐year‐old male was referred to our hospital for the evaluation of a mass‐like opacity visible on chest X‐ray. Computed tomography (CT) scan revealed a well‐defined soft tissue mass with internal low attenuations and adjacent multiple nodules in the upper lobe of the left lung, and surgical excision was subsequently performed. Histopathological findings revealed adipose tissue with lipoblasts and spindle cells and immunohistochemical staining (IHC) revealed the tumor cells were strongly positive for CDK4 and MDM2. Histopathological examination of the small lung nodules showed spindle cell proliferation and adipose tissue without positivity for MDM2. Here, we report a rare case of primary pleural liposarcoma combined adjacent spindle cell lipoma of the lung. Liposarcomas are soft tissue sarcomas that usually present in the extremities and retroperitoneum. Primary pleural liposarcoma is extremely rare and more common in males.A 66‐year‐old male was referred to our hospital for further evaluation of a lung mass, which was incidentally found on CT scan. He was an ex‐smoker and had no previous medical history. Physical examination showed no abnormalities in the chest and revealed stable vital signs. Chest X‐ray showed a mass‐like opacity in the lower zone of the left lung Fig . CT scanLiposarcoma accounts for about 20% of all mesenchymal tumors, is the most common soft tissue sarcoma in adults and is usually found in the retroperitoneum and the extremities.Primary pleural liposarcoma is extremely rare and the exact incidence is unknown. It is known to be more common in males and is primarily a disease of individuals of 50 years of age.MDM2, CDK4, and HMGA2.Liposarcomas originate from the primitive mesenchymal tissue residues rather than mature fat tissue. While the pleural cavity is formed from mesoderm, mesenchymal cells that are left in this process go through malignant transformation and bring about pleural liposarcoma.Spindle cell lipoma, which is composed of mature adipose tissue, collagen, and spindle cells is a rare benign tumor and commonly found in middle‐aged and older males.Intrathoracic spindle cell lipoma is usually diagnosed by histological examination. Microscopic appearance of spindle cell lipoma is characterized by the classic triad of mature adipocytes, ropey collagen bundles, and bland spindle cells. Spindle cell lipoma is strongly CD34 positive on immunohistochemical staining and the spindle cells may stain for S‐100.In conclusion, here we report a rare case of primary pleural liposarcoma combined spindle cell lipoma of the lung. This report might be helpful for the diagnosis of rare intrathoracic lipomatous tumors in the future.The authors declare that there are no conflicts of interest.


The conclusion derived from the information provided in this review is that disseminating tumor cells (DTC) collaborate with the microenvironment of a future metastatic organ site in the establishment of organ‐specific metastasis. We review the basic principles of site‐specific metastasis and the contribution of the cross talk between DTC and the microenvironment of metastatic sites (metastatic microenvironment [MME]) to the establishment of the organ‐specific premetastatic niche; the targeted migration of DTC to the endothelium of the future organ‐specific metastasis; the transmigration of DTC to this site and the seeding and colonization of DTC in their future MME. We also discuss the role played by DTC‐MME interactions on tumor dormancy and on the differential response of tumor cells residing in different MMEs to antitumor therapy. Finally, we summarize some studies dealing with the effects of the MME on a unique site‐specific metastasis—brain metastasis. ANGPTL4angiopoietin‐like 4BBBblood‐brain barrierBMPbone morphogenetic proteinCAMcell adhesion moleculesCLDN1claudin‐1CysCcystatin CDTCdisseminating tumor cellsECendothelial cellsEMTepithelial‐to‐mesenchymal transitionEVextracellular vesiclesHBBbeta‐subunit of human hemoglobinL1CAML1 cell adhesion moleculeMMEmetastatic microenvironmentPMMEpremetastatic microenvironmentTRPA1transient receptor potential ankyrin 1VCAM‐1vascular cell adhesion molecule 1XISTX‐inactive specific transcript1Stephen Paget is the conceptual father of the concept that metastasis is site‐specific. In his description of the “seed and soil” theoryThe major research thrust on site‐specific metastasis is directed toward several targets: defining the molecular signature of cancer cells that enable their establishment as metastatic lesions in different organs; the molecular signature of the host microenvironmental cells supporting or inhibiting this establishment; the modus operandi by which such molecules exert their pro‐ or antimetastasis functions; the functional significance of interactions between metastasizing cancer cells with cells residing in or recruited to the microenvironment of specific organ sites and the impact of site‐specific metastasis on and response to therapy. The following references encompass a partial list of recent reviews on cancer metastasis that alluded to organ specificity.This review will summarize the principles governing organ‐specific metastasis and focus on interactions between cellular and molecular components of the metastatic microenvironment (MME) of specific organs with cancer cells infiltrating into the microenvironment of these organs.The microenvironment of an organ belonging to a tumor bearer without evidence of metastasis in this organ will be designated as premetastatic microenvironment (PMME). The microenvironment of the same organ harboring micro‐ or macrometastasis will be designated as MME.2It is well established that tumor cells interact by multiple mechanisms with cellular and molecular components of their microenvironment. These interactions reprogram and shape the phenotype of both interaction partners. In certain cases, such interactions may lead to metastasis.Different cancer types usually metastasize to multiple but favored organ sites. Breast cancer for example, metastasizes to bone, lungs, liver and brain while prostate cancer metastasizes primarily to lymph nodes and to bones.The major determinants of site‐specific metastasis are alterations in the genetic, epigenetic and proteomic profiles of metastasizing cancer cells as well as factors derived from the MME.Although tumor cells reach the vasculature of all organs, metastasis develops only in selected organs. Specific adhesive recognition between cancer cells and the endothelium of the target organ, as well as nonspecific anatomical or mechanical factors such as circulation mechanics are involved in the arrest of circulating cancer cells at the target organ.The ability of metastasizing tumor cells to survive and propagate at a specific organ site, being an essential prerequisite for metastasis formation, is enabled to a large extent by MME‐derived survival and growth factors.The concept of site‐specific metastasis lay dormant until the 1980s. The awakening of this concept revealed the basic principles of site‐specific metastasis as we know them today.With these issues in mind, it is essential to be aware of the possibility that many, perhaps most, events related to tumor progression in general, occur simultaneously and that most such events may yield Yin‐Yang results.This review will focus on the involvement of the MME in the four major phases of site‐specific metastasis, namely creation of the premetastatic niche; migration of metastasizing tumor cells to specific organ sites; invasion of the tumor cells into the target organ and the establishment of dormant or overt metastasis in this organ.3Kaplan and coworkers were the first to demonstrate that future metastatic sites are preconditioned for colonization of circulating tumor cells.The initial step of premetastatic niche formation in specific organ sites is mediated by tumor‐derived soluble factors such as TNF‐α, TGF‐β, CXCL12, placental growth factor and VEGF‐AThe recruited cells play very important functions in creating, in a foreign milieu, a hospitable microenvironment for incoming tumor cells and for their progression toward metastasisThe cargo of exosomes contains single‐ or double‐stranded DNA, mRNA and noncoding RNAs, proteins and various types of glycans. All these molecules, directly or indirectly exert numerous and varied effects on the PMME, MME and tumor cells alike. For example, exosomes increase vascular leakiness, reprogram the ECM, suppress anti‐tumor immune reactivity and promote tumor progression.One of the most important and relevant issues regarding premetastatic niche formation (and to the topic of this review) is the mechanism by which tumor‐derived exosomes locate the “correct address” of the organ, which will serve as the future home for metastasis originating in the corresponding tumor. In other words, what are the specific recognition units expressed on the surface of exosomes that mediate the interaction with the target organ via complementary ligands and what are these ligands?Several molecular mechanisms may underlie the organotropism of tumor‐derived exosomes (and the subsequent formation of metastasis specific to a particular organ).The most extensive analysis of the organotropism of tumor‐derived exosomes was performed by Hoshino and collaboratorsIn view of the above, it is not unlikely that integrin‐integrin ligand axes are the major mechanisms responsible for the organotropism of tumor‐derived exosomes and for site‐specific metastasis. In order to prepare tailor‐made therapy modalities for metastasis lodging in different organs, it is essential to fully assess the unique molecular and pathological processes that may occur in each MME before the development of metastases.4Tumor cells disseminating from the primary tumor (disseminating tumor cells—DTC) reach secondary organ sites by employing two major, nonmutually exclusive mechanisms. The one, triggered by the TME, involves the reprograming of adhesive epithelial cancer cells to migratory and invasive mesenchymal cells . This general mechanism may culminate in metastasis formation.The machinery of targeted migration employed by inflammatory leukocytes to reach inflammatory sitesA study from our laboratory established that the chemokine receptor CCR4 is a member of the molecular signature of melanoma brain metastasis and that its chemokine ligands CCL17 and CCL22 are expressed in microenvironmental brain cells.In addition to directing tumor cells to specific organ sites via “cellular highways,” chemokine‐chemokine receptor axes exert other metastasis‐facilitation functions such as promotion of tumor proliferation and of angiogenesis as well as recruitment of suppressor immunocytes.55.1In addition to the entrapment of DTC in small capillaries of most organs,Pericytes are involved in tumor progression toward metastasis.ECs of one particular organ often express a different profile of cell surface proteins compared to ECs of other organs. This organ specificity accounts for the fact that DTC bind specifically to the vasculature of some organs but not to that of others.Leukocyte extravasation is a multistep process whereby leukocytes expressing fucosylated selectin ligands role over cytokine‐activated selectin‐expressing ECs. Subsequently the leukocytes encounter chemokines expressed on the surface of ECs. This encounter triggers the activation of leukocyte‐expressed integrins and subsequently a firm adhesion to the activated endothelium. Eventually, diapedesis regulated by the activation of Rho GTPases takes place.The complex extravasation cascade of leukocytes during inflammatory responses was co‐opted (some would use the term hijacked) by circulating cancer cells. This co‐option is manifested inter alia by the utilization of surface molecules involved in leukocyte extravasation also for endothelial transmigration of DTC.Several cell surface molecules including integrins, selectins, other cell adhesion molecules, glycosyl transferases, glycolipids and fetal recognition systemsThe co‐aggregation of DTC with neutrophils5.2Early studies demonstrating the organ‐specific nature of DTC‐EC binding were performed by Auerbach et al.Several adhesion molecules expressed by DTC or by endothelia of specific organs are determinants of site‐specific metastasis. Molecular determinants and mechanisms mediating adhesion of DTC to EC and transendothelial migration were characterized with respect to lymph nodes,66.1Disseminated tumor cells endowed with a genetic repertoire that sanctions their colonization in the PMME of a specific organA case in point is the production of reactive oxygen and nitrogen species either by DTC themselves or by the PMME/MME. The exposure of the metastasizing cancer cells to the oxygen/nitrogen‐mediated cytotoxic insults may lead either to the survival of highly resistant tumor variants and/or to the elimination of sensitive ones.In general, ECM, the vascular system, immunity, metabolism and other cellular and molecular constituents of the PMME/MME engage in a cross talk with DTC leading to their colonization in various organ sites.In the following, there are several examples for tumor‐promoting mechanisms that are not selective for specific DTC/MME combinations.The contribution of fibroblasts residing in malignant tumors (cancer‐associated fibroblasts—CAFs) to cancer progression toward metastasis has been thoroughly studied.Hepatic stellate cells are transformed by colorectal cancer‐derived signals to fibroblasts. These hepatic fibroblasts express CCL12, thereby contributing to the targeted migration of CXCR4‐expressing cancer cells to the liver.Macrophages in the premetastatic niche promote tumor invasion into the niche and subsequently take part in the generation of metastasis. Macrophages within tumor/metastatic lesions (tumor‐associated macrophages—TAMs) promote propagation of tumor cells, induce matrix remodeling and angiogenesis, and establish an immunosuppressive microenvironmentNeurogenesis is a determinant in the seeding/colonization of tumor cells in the MME.The L1 cell adhesion molecule (L1CAM) is used by DTC to spread on capillaries of the PMME/MME. The spreading leads to activation of Yes‐associated protein (YAP) being a necessary component of colonization of various types of cancer in different organs.Essentially all cancer types evoke inflammatory reactions and antitumor immunity at the microenvironment of the primary tumor as well as in the MME of essentially all organ sites. These responses may either promote or restrain tumor progression.6.2The seeding of infiltrating tumor cells in the PMME and their subsequent colonization and progression in specific organ sites were summarized in several reviews.As mentioned earlier, DTC that succeeded to complete the migratory journey through the endothelium of “preferred” specific organs need the support of the PMME (and subsequently the MME) in order to survive, proliferate and overcome unfavorable and hostile microenvironmental factors.Below are several examples of site‐specific colonization of DTC. Neurogenesis, angiogenesis and various growth factors take part in the poststroke tissue repair process, which leads to the regeneration of new blood vessels and new neurons. In a recent study, we demonstrated that melanoma cells utilized (hijacked) factors in the poststroke regenerative neurovascular niche to form brain metastasis. Specifically, we demonstrated that brain microenvironmental cells such as ECs or astrocytes from the peri‐infarct region of the brain or cells that were subjected in vitro to stroke‐inducting conditions (oxygen and glucose deprivation) secreted factors that promoted brain metastasis formation by melanoma cells.VCAM‐1 expressed by disseminating breast cancer cells binds to α4‐integrin expressed by metastasis‐associated macrophages. This binding activates Akt signaling in lung‐metastasizing cancer cells, thereby protecting them from apoptosis.Propagation of invading cancer cells can be promoted by both tumor‐derived factors and those emitted from the PMME/MME. For example, extracellular vesicles secreted from highly metastatic clones of a human osteosarcoma are capable of inducing a high metastasizing behavior on poorly metastatic clones from the same tumor.The mechanisms by which immunity is involved in resistance against tumor and metastasis formation, propagation and progression are, in most cases, uniform for most cancer types.Infiltrating myeloid‐derived cells promoted the formation of breast cancer metastases in the liver but did not affect the formation of lung and bone metastasis. Depleting the granulocytic component of the infiltrate resulted in significantly impaired formation of liver metastases. Neutrophils that infiltrated the liver metastases expressed the malignancy enhancing N2 phenotype.CXCL12 and TRAIL expressed in the MME of the boneregulate Akt signaling and survival responses in bone‐metastasizing breast cancer cells,IL‐1β‐expressing prostate cancer cells generated a higher load of bone metastasis than prostate cancer cells that do not express this cytokine. Moreover, IL‐1β‐expressing cells created an MME that supported bone metastasis formation also by prostate cancer cells that do not express IL‐1β. This suggested that IL‐1β may promote bone colonization by prostate cancer.3CL1‐CX3CR1) plays an important regulatory role in organ‐specific peritoneal colonization of ovarian cancer. A high expression of CX3CR1 by the tumor cells correlates with a highly malignant phenotype while its downregulation reduces its interaction with CX3CL1 expressed by human mesothelium and reduces metastasis.The fractalkine axis , or as single cells or cell clusters that do not grow due to an equal rate of proliferation and cell death (mass dormancy)Dormant micrometastatic tumor cells reside in various MMEs alongside with frank metastases.Several studies indicated that the specific organ MME dictates dormancy of DTC and that dormancy functions as a survival strategy of such cells when their progression toward frank metastasis is blocked due to a variety of reasons.Dormancy induction of bone‐metastasizing prostate cancer cells represents a typical case of MME‐regulated dormancy at a specific organ site. TGFβ2,The concept of “Microenvironmental Control” was independently proposed by Klein and KleinThe MME plays an important role also in mechanisms that awaken dormant micrometastatic tumor cells.8Primary and metastatic tumor cells and their microenvironments control each other's phenotype.Do metastatic tumor cells residing in a particular MME respond to anticancer therapy similarly or differently than tumor cells  residing in a different MME? This question is obviously of high clinical relevance.A meta‐analysis of response of breast cancer metastasis to various treatment modalities in relation to metastatic sites revealed that HR‐positive metastases located in different organs and subjected to certain forms of targeted therapy responded similarly. On the other hand, hormonal therapy as well as HER2‐targeted therapy were more effective in visceral metastases than in nonvisceral metastases.Several studies that dealt with the response of metastasis in different MMEs concluded that metastasis lodging in diverse environments exhibit a differential responsiveness to cancer therapy.In breast cancer, RANKL and Jagged1‐targeting molecules as well as EGFR inhibitors reduce bone metastasis. Antibodies directed against the astrocyte‐elevated gene‐1 protein (encoded by the MTDH gene) diminish lung metastasis and various anti HER2 agents delay brain metastasis.In order to improve the efficiency and success of both current and future treatment modalities of metastatic diseases, the issue of differential responses of metastatic cells inhabiting different MMEs should be thoroughly investigated. Precision medicine should include the introduction of tailor‐made therapies for cancer cells that metastasize to distinct MMEs.9“The brain is a complex biological organ of great computational capability that constructs our sensory experiences, regulates our thoughts and emotions, and control our actions”.Eric KandelThe brain differs from other organs in many parameters including but not limited to a unique anatomy, cellular heterogeneity and molecular milieu. All these factors create a supercomplex network of signaling pathways.Metastasis to the brain constitutes a major unmet clinical challenge.In addition to metastasis genes that determine the formation of brain metastasis,It is important to note that many of such interactions take place simultaneously and may be affected by factors from compartments external to the brain. This calls for caution in the interpretation of results derived from analyzing tumor‐brain cross talk. Here, we summarize several instances of tumor‐brain cross talk and their biologic consequences. We will include studies from our lab focusing on melanoma brain metastasis. Neurogenesis plays an important role in metastasis formation of several tumors (see later) and obviously also in brain metastasis.The enhanced infiltration of the melanoma cells through the BBB endorses brain metastasis.Neural repair after stroke involves neurogenesis among other proliferative programs. A recent report indicated that regenerating neurogenesis induced by stroke promoted brain melanoma metastasis.The BBB structure composed of ECs, pericytes and astrocytes prevents the nonselective entry of unwanted particles and molecules into the CNS.9.1The specific features and properties of the BBB  distinguish it from vasculatures of other organs.As pointed out above adhesion of tumor cells to endothelium is the first step of transendothelial migration. Below is a summary of several studies that characterized molecules and signaling pathways involved in the adhesion of cancer cells to brain endothelium and in their transendothelial migration to form brain metastasis. An early study revealed that TNF or IL‐1 promoted the adhesion of murine tumor cells to brain endothelium.Tight junctions play an important role in sustaining the barrier function of the brain endothelium.Results from our lab revealed a set of observations that, at first glance, seemed unrelated. Human melanoma brain metastasis variantsRho‐associated protein kinase (ROCK) signaling pathway is involved in the interaction of metastatic melanoma cells with the brain endothelium. The inhibition of the kinase augmented the adhesion of the melanoma cells to endothelium, increased their transendothelial migration and promoted the formation of brain metastasis.The The BBB of brain metastasis is leaky.The BBB produces a variety of cytokines and other factors such as extracellular vesicles and responds to factors produced by tumor cells or by other brain microenvironmental cells. Such factors may have a far reaching, however opposing, impact on the capacity to form brain metastasis.9.2Although the physiological and some pathological functions of pericytes (including their role in metastasis) have been reviewed,Pericytes regulate vascularization of brain metastasis by being the main source of the connective tissue in such metastasis.The formation of brain metastasis by mouse breast cancer cells is associated with an alteration in the representation of pericyte subpopulations. An increase of desmin‐expressing pericytes and a decrease of CD13‐expressing pericytes in the brain is associated with increased permeability of the barrier separating breast cancer brain metastasis from the circulation.Brain pericytes exert an antimetastatic effect by regulating BBB resistance to the formation of lung cancer brain metastasis. Conditioned medium of pericytes suppressed the proliferation of the cancer cells.9.3The complex roles of astrocytes in brain metastasisA study demonstrating that astrocytes exert antibrain metastatic activity showed that astrocytes in breast and lung brain‐metastasis secrete plasmin, which cleaves FasL expressed by astrocytes, thereby generating a death signal for the metastatic cells. Plasmin also inactivated L1CAM expressed by the metastatic cells, which was utilized by them as a spreading factor. The antimetastatic function of astrocyte‐derived plasmin was counteracted by cancer cell‐derived serpin.Transient receptor potential ankyrin 1 (TRPA1) is an ion channel that drives brain metastasis of lung cancer by activating fibroblast growth factor receptor 2 (FGFR2). Astrocytes block metastasis by downregulating TRPA1 via miRNA‐142‐3p.Below are some recent reports on the prometastatic functions of astrocytes and on the molecular mechanisms by which these brain cells exert their promoting activities on brain‐metastasizing cancer cells.Astrocytes promote the development of brain metastasis by upregulating the expression of survival genes and by activating tumor promoting pathways in brain metastasizing cells.Extracellular vesicles from brain‐metastasizing melanoma cells activate proinflammatory signaling pathways in astrocytes.Two studies from our lab provided additional proof for the support provided by astrocytes to the establishment of melanoma brain metastasis. Human brain‐metastasizing melanoma cells induced the expression by and secretion of IL‐23 from astrocytes. This cytokine, in turn, reciprocally upregulated the secretion of MMP2 from the metastatic melanoma cells, thereby enhancing their invasiveness.9.4“As the immune‐competent cells of the brain, microglia play an increasingly important role in maintaining normal brain function.”Similarly to astrocytes, microglia may both support and restrain brain metastasis.In a study performed in our lab,As with studies on the effect of astrocytes on the formation and maintenance of brain metastasis, studies reporting antimetastatic effects of microglia are less numerous than those reporting the opposite. Below are some examples of studies on anti‐ or pro‐brain metastasis functions of microglia.Systemic prophylactic administration of CpG‐C, a TLR9 agonist, reduced brain seeding and colonization of mouse and human lung cancer and of mouse melanoma.Employing a mouse model system, it was found that activated microglia lysed murine and human tumor cells by release of nitric oxide. Activated microglia did not lyse non tumorigenic control cells.A review of the cross talk between microglia and melanoma brain metastasisPI3K was identified as a master regulator of metastasis‐promoting microglia. Treating brain‐metastasizing mouse mammary tumor cells with a pan‐PI3K inhibitor suppressed significantly metastasis formation by the tumor cells. These results suggest that PI3K inhibition may be developed into a brain metastasis therapy modality.Simultaneously with the development of B16 melanoma brain “metastasis” , neighboring microglia became increasingly activated, gradually accumulating in the metastatic lesion and thereby promoting further growth of the lesion. Brain “metastasis” was inhibited by microglia depletion in vivo and microglia‐derived MMP3 seemed to facilitate melanoma cell growth.A study from our lab indicated that the secretion of cystatin C (CysC), an extracellular cysteine protease inhibitor, from brain‐metastasizing melanoma cells as well as from microglia is increased after these two cell types engaged in an in vitro cross talk. CysC enhanced the migration of melanoma cells through an in vitro model of BBB and promoted the formation of melanoma three‐dimensional structures in matrigel. Immunohistochemistry (IHC) demonstrated high expression levels of CysC in the brain of nude mice bearing xenografted human melanoma brain metastasis. These results indicate that CysC promotes melanoma brain metastasis.10Scores of published studies identified factors in the tumor microenvironment that either promote or restrain metastasis, the final stop of tumor progression.Are all these factors or only certain combinations thereof necessary in order to generate metastasis? In other words, are we able to separate the wheat from the chaff and to identify the most relevant factors that generate, sustain or resist metastasis? Are there several redundant, nonmutually exclusive signaling pathways that have the capacity to inhibit or promote metastasis? For example, a road block posed by an anticancer drug neutralizing a prometastasis pathway may be circumvented by another, redundant, prometastasis pathway, thereby allowing uninterrupted tumor progression.Providing answers to the above questions (and to numerous related ones) might enable the construction of a comprehensive, overall big picture of tumor progression toward metastasis. This is a major and challenging task for the metastasis research community in efforts to develop and apply drugs that will prevent metastasis or cure it.To meet this goal, we have to abandon, as far as possible, reductionism and to employ approaches used in analysis of big data, in Bioinformatics and in Systems Biology.The authors declare that they have no conflict of interest.


DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approaches for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. We provide examples of repair in DNA nanostructures showing the difference between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples. Self‐assembly and reconfigurability of DNA origami is exploited to realize self‐repairing functional nanodevices. A self‐regenerating brightness label is realized by constant exchange of labeling strands from solution. Structural self‐healing of a DNA origami nanoruler is achieved by excess of staple strands that exchange with enzymatically or photo‐induced damaged strands. A molecular machine after Stoddart is defined as the assembly of a controlled number of molecular building units, that is designed to perform controlled motions as the output for an external stimulation (input).Fundamentally, molecular devices are prone to rapid degradation and loss of functionality due to the high proportion of surface atoms and molecules.For applications of functional DNA nanotechnology, research has focused mainly on the improvement of stabilization of DNA nanostructures in complex environments, for example, by coating or encapsulation of the structure or strengthening the backbone by covalent cross‐linking.21, 22Here, we propose to exploit the self‐assembly and reconfiguration abilities of DNA origami technique to introduce general mechanisms for self‐repair within defective or externally damaged nanostructures.26, 27−2), the DNA origamis photobleach  of the same imaging area to see whether the brightness rulers recover in the presence of the 5 nM solution of imager strands but only observed a small recovery of the fluorescence  had to be applied to achieve the complete bleaching of the self‐regenerating labels  containing two spots with three docking strands per spot for DNA PAINT imaging experiments . We then added staple strands with DNA PAINT docking strand extensions that would form a third spot on the DNA origami for DNA PAINT binding studies when incorporated. Efficient incorporation of the added staples could only be observed when the NRO nanorulers were previously assembled with shorter staple strands so that a toehold of 4 or 8 nucleotides was formed within the scaffold strand  confirming the notion that a toehold is required for efficient strand displacement reactions also within an intact DNA origami.Previous work showed the rapid degradation of unmodified DNA nanostructures in 10 % FBS solution within 24 h.45, 46Nb.BtsI to the labeled nanorulers that specifically hydrolyses the imager strand exactly in the middle when it is hybridized to the docking site so that single‐stranded imager strands in solution stay intact. The resulting two 10 nt fragments are not stably bound to the DNA origami and dissociate rapidly leaving a brightness ruler with strongly reduced fluorescence signal  and with a solution of 5 nM imager strands and Nb.BtsI (H) . Extracted averaged intensity transients over hundreds of nanorulers under time‐lapse imaging  are given in Figure Nb.BtsI to externally labeled 6HB brightness ruler led to accelerated loss of brightness due to enzymatic cleavage. After about 2 h a plateau below 20 % of the initial fluorescence intensity was reached. When the Nb.Btsl and a 5 nM imager strand solution were added simultaneously, no degradation was visible over 1200 min of time‐lapse imaging showing that self‐repair mechanisms can quantitatively compensate mechanisms of wear out , active self‐healing and self‐regeneration mechanisms outlined here provide general strategies to address random and unknown damage. This is best illustrated by drastic improvement in the stability of DNA nanostructures in a complex and chemically demanding FBS environment shown in Figure In the development of materials and molecular machines with increasing complexity, their robustness and their resistance against wear as well as their ability to prevail in complex environments call for new approaches of protection. In this context, self‐repair mechanisms that are common in nature also become more important to be implemented in artificial systems. As DNA nanotechnology enables self‐assembling nanostructures and molecular functional devices of highest complexity, we exploit self‐assembling and reconfiguration properties to implement self‐repair mechanisms. These include self‐regeneration by a pool of intact building blocks and exchange under conditions of thermal equilibration as well as more specific self‐healing that only allows exchange of building blocks upon occurrence of a damage. We showed that such mechanisms can already be implemented in existing applications of DNA origami nanorulers and brightness references. Self‐repair strategies might become a crucial area of research when pursuing our visions of sustainable, long‐lasting molecular nanorobots.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Understanding the nature of biological invasions is an important grand challenge, and we share Prof. Hedrick's concern for developing good theory and mechanistic understanding of the ecological and evolutionary dynamics of invasion Hedrick, .FIS cannot be calculated from the expected and observed heterozygosity values provided in the table 1 of Winkler et al.  is the ith allele frequency of the lth locus in the gth subpopulation, or the average expected proportion of heterozygote individuals within subpopulations . We encourage others to utilize these data and hope our contributions advance our understanding of the biological mechanisms enabling species like Sahara mustard to invade with the speed and force we have witnessed in recent decades across the US Southwest.We have confidence in the sequence data used in analyses as they were quality‐controlled through standard, rigorous checks. Sequencing was carried out on an Illumina HiSeq2000 platform at the University of Oregon, and nextRAD methodology is detailed in Russello et al. . Prior tWe should also note that the biology of novel species invasion might reveal patterns that are unexpected given traditional approaches, as exploring new phenomena can challenge the assumptions of the techniques we have used to study population dynamics. For example, we show Sahara mustard has been introduced at least three separate times in the United States, each of which have either spread beyond their initial introduction sites or remain isolated, at least up to the season we sampled individuals ; Project administration (lead); Writing‐original draft (lead). Kenneth James Chapin: Methodology (supporting); Writing‐review & editing (supporting). J. David Garmon: Methodology (supporting); Writing‐review & editing (supporting). Brandon S. Gaut: Methodology (supporting); Writing‐review & editing (supporting). Travis E Huxman: Methodology (supporting); Writing‐review & editing (supporting).B. tournefortii: PRJNA534338).Molecular data used in these analyses are available as a NCBI’s sequence read archive (BioProject for


Genotyping‐by‐sequencing has enabled approaches for genomic selection to improve yield, stress resistance and nutritional value. More and more resource studies are emerging providing 1000 and more genotypes and millions of SNPs for one species covering a hitherto inaccessible intraspecific genetic variation. The larger the databases are growing, the better statistical approaches for genomic selection will be available. However, there are clear limitations on the statistical but also on the biological part. Intraspecific genetic variation is able to explain a high proportion of the phenotypes, but a large part of phenotypic plasticity also stems from environmentally driven transcriptional, post‐transcriptional, translational, post‐translational, epigenetic and metabolic regulation. Moreover, regulation of the same gene can have different phenotypic outputs in different environments. Consequently, to explain and understand environment‐dependent phenotypic plasticity based on the available genotype variation we have to integrate the analysis of further molecular levels reflecting the complete information flow from the gene to metabolism to phenotype. Interestingly, metabolomics platforms are already more cost‐effective than NGS platforms and are decisive for the prediction of nutritional value or stress resistance. Here, we propose three fundamental pillars for future breeding strategies in the framework of Green Systems Biology: (i) combining genome selection with environment‐dependent PANOMICS analysis and deep learning to improve prediction accuracy for marker‐dependent trait performance; (ii) PANOMICS resolution at subtissue, cellular and subcellular level provides information about fundamental functions of selected markers; (iii) combining PANOMICS with genome editing and speed breeding tools to accelerate and enhance large‐scale functional validation of trait‐specific precision breeding. It is well estimated that by 2050, the world population will reach ~9 billion and ~9–11 billion by 2100. The current practice of food production using conventional breeding approaches is insufficient to satisfy the global demand by 2050, especially with the view to agroecological questions such as productivity, robustness, sustainability and equitability. Moreover, agricultural productivity and environmental changes are well correlated; any abrupt change in environmental conditions will have a consequent harsh impact on plant productivity owing to the direct and indirect impact of biotic and abiotic stresses , leaf features , carbon use efficiency (CUE), nitrogen use efficiency (NUE) and water use efficiency (WUE) determine major traits that contribute for overall crop performance and yield Figure . For exaArabidopsis thaliana. However, such a detailed understanding of crop plants is rather at its infancy.Opportunities for crop improvement include engineering of these complex traits to improve yield. This requires a thorough understanding of the genetic system and information flow from gene, RNA, protein and metabolite to these traits. Furthermore, developing ecophysiological models by integrating the physiological traits with the PANOMICS approach can improve prediction accuracy and marker identification in the environmental context. Considerable progress has been made in deciphering the genetic and molecular basis of the developmental processes that govern these traits, particularly in the model plant species et al., et al., The application of genomics in the field of plant breeding started with the advent of RFLP marker technology  to enhance breeding lines and (b) improve genetic stocks. Genetic stocks represent new types of germplasm collections that include mutants generated using DNA insertion . This information improves the understanding of causal processes and the prediction accuracy for genomic selection  sequencing that can enable a clear understanding of distinct cell identities and transition states and it also provides evidence about the unique mutations in the cell , subcellular localization and compartmentalization, protein complexes, signalling pathways and protein–protein interactions, all this not predictable from the genome sequence  often leads to delayed seed germination, reduced plant growth and decreased crop yield. The role of proteins in plant stress response is crucial, since proteins are directly involved in shaping novel phenotype by adjusting physiological traits to the altered environment  with metabolomics has emerged as a powerful forward genetics strategy to dissect the genetic and biochemical bases of crop plants Luo, . UtilizaArabidopsis thaliana to identify the effects of widespread genetic variants on metabolic diversity across natural populations . However, two main questions still remain open in the mGWAS analysis (i) How to dissect metabolite pathways and characterize gene function in post‐transcription, post‐translation and epigenetic condition? (ii) mGWAS is based on the ratio of each individual metabolite under different conditions, is this approach dynamic enough to dissect mechanisms underlying metabolic responses to environmental stimuli?et al., The phenotypic outcome depends upon single or multiple genes and their interaction with the environment. Phenomics is the systematic study that involves the gathering of phenotypic data from multiple levels of the organization and progress towards the more complete characterization of the holistic phenotype  ; Phenome Networks database (http://phnserver.phenome‐networks.com/); PHOTOSYNQ (https://photosynq.org/); International plant phenotyping network (IPPN) (https://www.plant‐phenotyping.org/IPPN_home); and European Infrastructure for Multi‐scale Plant Phenomics and Simulation (EMPHASIS) (https://emphasis.plant‐phenotyping.eu/). However, in the current scenario, phenotypic data should be cross‐referenced to the other plant resources such as gene banks, specific genomic information, syntenic, genetic and physical maps, SNP marker, metabolomics and proteomics databases. Eventually, such cross‐reference between phenotype and genomic information can be exploited for crop improvement. Moreover, integrating speed breeding platforms  is majorly based on remote sensing; it is a nondestructive and noninvasive approach. It is based on the principle of gathering information provided by visible/near‐infrared and far‐infrared radiation emitted by the crops. It is the most commonly used approaches in crop phenotyping. The devices used for phenotyping include multispectral, hyperspectral, fluorescence, thermal sensors and imagers (RGB colour cameras). Extensive literature is available with detailed information regarding the use of these devices and advancements in field phenotyping  in Spain to track photosynthesis at different phenological and stress stages throughout the season and to support its application in the context of precision breeding  and generate models that can be used to predict complex traits  is gaining momentum in multi‐omics data integration . However, these mapping populations are products from few cycles of recombination events, limiting the resolution of genetic maps that often do not represent germplasm that is actively used in breeding programmes. By contrast, GWAS overcome these limitations and provide greater resolution for identifying genes potentially responsible for variation in a quantitative trait  in plants, it is of utmost importance to understand and gather information about both the specific loci that underlie a phenotype, and the genetic architecture of a trait  with the identification of ~3.6 million SNPs and phenotyped 14 agronomic traits. Interestingly, the obtained result only explained ~36% of phenotypic variances and the complex genetic architecture of these traits , gene knockouts, activation or repression of genes and epigenetic changes are possible . Several public institutes such as ICRISAT , which belongs to the domain of CGIAR , have been accelerating use of genome editing tools  for enhancing crop production and support smallholder farmers (https://www.icrisat.org/icrisat‐and‐corteva‐agriscience‐agriculture‐division‐of‐dowdupont‐collaborate‐for‐sharing‐advanced‐breeding‐technologies‐to‐improve‐crops‐that‐feed‐millions/). This kind of initiative will accelerate the process of bringing healthier legumes (such as chickpea and pigeon pea) to the consumers  will allow one to perform molecular characterization of genotypes, providing the list of candidate genes/gene products that are highly valuable for breeding and engineering stress‐tolerant crops with novel and valuable traits not reachable by classical genome prediction methods. Still, proteomics and metabolomics studies are often regarded as holistic studies due to the fact that not many putative markers are translated into the productive sector. This is because high‐throughput techniques are still emerging and require steady improvement in instrumentation and algorithms. The cost of generating high‐throughput data needs to decrease substantially because it is important to identify relevant genotype‐phenotype associations that are not predictable from the genome sequence. However, metabolomics platforms are nowadays partially cheaper than NGS platforms and have a higher throughput. Metabolite analysis is very important to understand, for example nutritional value of crops, but also stress resistance. Accordingly, the metabolic readout can be a rapid predictor of important traits of large cohorts of samples. Here, we propose the complementation with PANOMICS because these technologies become more and more cost‐effective and will improve genomic prediction. Eventually, the integration of PANOMICS platforms with systems modelling and genome editing techniques will enhance precision breeding and support quick breeding procedures (such as SPEED breeding) with the ultimate outcome of providing the appropriate cultivars for each agroecological scenario.The authors declare no competing financial interest.WW, PC, AG and RKV conceived the conception of the manuscript; PC, AG and WW drafted the manuscript; RKV critically reviewed the manuscript; AG and AB prepared data tables; PC and AG designed the figures. All authors have revised and approved the final version of the manuscript.


High‐throughput identification technologies provide efficient tools for understanding the ecology and functioning of microorganisms. Yet, these methods have been only rarely used for monitoring and testing ecological hypotheses in plant pathogens and pests in spite of their immense importance in agriculture, forestry and plant community dynamics. The main objectives of this manuscript are the following: (a) to provide a comprehensive overview about the state‐of‐the‐art high‐throughput quantification and molecular identification methods used to address population dynamics, community ecology and host associations of microorganisms, with a specific focus on antagonists such as pathogens, viruses and pests; (b) to compile available information and provide recommendations about specific protocols and workable primers for bacteria, fungi, oomycetes and insect pests; and (c) to provide examples of novel methods used in other microbiological disciplines that are of great potential use for testing specific biological hypotheses related to pathology. Finally, we evaluate the overall perspectives of the state‐of‐the‐art and still evolving methods for diagnostics and population‐ and community‐level ecological research of pathogens and pests. For spiking, known quantity of control DNA or individuals is added to the sample prior to DNA/RNA extraction and the quantity of target organisms or genes is detected based on the relative amount of obtained sequences  than Sanger sequencing, and the analysis chemistry was rapidly optimized to provide high‐quality reads from 50 to 700–1,000 bases at 1.2 million read throughput , was developed in early 2000s. The 454 technology was >100‐fold cheaper (10www.illumina.com) and Ion Torrent (www.iontorrent.com) technologies replaced 454 in the early 2010s because of greater throughput at lower costs. Nonetheless, the Ion Torrent is continuously haunted by short read length (up to 450 bp) and fluctuating sequence quality, which has limited its use in analysis of soil and plant samples , which is similar to the Illumina platform, was released. So far, it has been used for metagenomic detection of human pathogens  and Oxford Nanopore (www.nanoporetech.com) technologies, which became commercially available in 2011 and 2015, respectively. However, both of these platforms have very high initial error rates (10%–15% per base) that have improved only marginally in the last years. In the PacBio platform, circularized DNA molecules are sequenced multiple times, reducing the error rate to a minimum (0.1%) at 9‐ to 11‐fold consensus  compared with Illumina sequencing. Thus, PacBio is the method of choice for metabarcodes >550 bp given sufficient funding.As a major technical advance, much longer DNA fragments spanning tens of kilobases can be sequenced using the Pacific Biosciences  and Intramolecular‐ligated Nanopore Consensus Sequencing (INC‐Seq) technique  Indian origin for the pathogenic strains. Nanopore consensus sequences have been used to acquire long DNA barcodes from multiple arthropod specimens simultaneously . Several examples of using RAD‐seq in plant pathogens are given in Grünwald et al.  is the generation of several orders of magnitude more information about polymorphic sites and a better understanding of their linkage and occurrence in exons and introns  DNA fragments may be preserved for millennia in anoxic sediments and permafrost  is difficult to perform in natural conditions because of multiple carbon sources diluting the isotopic signal. This is especially relevant for eukaryotic pathogens that grow and accumulate 13C or nucleotide analogues into their DNA slowly and may use much of the labelled carbon for respiration. Nonetheless, 13C incorporated into DNA and fatty acids revealed flow of plant‐derived carbon through the soil food web and decline in pathogen‐to‐mycorrhiza ratio during secondary succession  and these short fragments prime the templates during subsequent cycles. Chimeric molecules develop more commonly between two closely related organisms becoming more abundant with increasing number of PCR cycles and community complexity  are small and expected to represent the community poorly, pooling multiple samples is a viable option. In case of hierarchical design , it is useful to pool multiple subsamples when the internal variation is not of interest. However, in most other cases, analysis of multiple independent samples is preferable due to the ability to estimate sampling error and address the importance of spatiotemporal variability. HTS analyses can easily recover slight shifts in taxonomic and gene composition; therefore, multivariate techniques require just 3–4 replicates to detect biologically important shifts in community composition  and QIIME (www.qiime.org), are run on command line. Both of these popular bioinformatics platforms are optimized for alignment‐based bacterial 16S rRNA gene analysis. QIITA (https://qiita.microbio.me) is a recently developed web‐based analysis platform for bioinformatics and analysis workflow optimized for bacteria‐targeted and metagenomic research. PipeCraft is a user‐friendly software with a graphical interface, multiple options incorporated from other programs, capacity to analyse metabarcoding data from all sequencing platforms and compatibility with Linux, Mac and Windows, which all attract non‐bioinformatician users  may precede or follow chimera check. Extraction of ITS and other variable regions in rRNA genes enables simultaneous removal of non‐target organisms and focus on a shorter but more variable barcode that has improved taxonomic resolution  is too conservative for species‐level identification of most taxa. For example, some biological species of All clustering methods generate more OTUs than expected at any barcoding threshold with increasing sequencing depth, indicating accumulation of PCR and sequencing errors into rare “satellite” taxa , SILVA (www.arb-silva.de) and UniEuk (https://unieuk.org/) have generated databases and reference data sets populated with filtered and third‐party annotated sequences. SILVA is focused on nuclear SSU and LSU sequences of prokaryotes and eukaryotes, but both oomycetes and fungi are poorly represented and have problems with taxonomic assignment  in addition. Animal and human pathogens have annotated sequence data in the ISHAM‐ITS database  linked to Digital Object Identifiers (DOIs). These machine‐readable DOIs enable communication of both named and unnamed taxa across studies and time , or use these functions as covariates in uni‐ and multivariate statistics . Information about the OTUs in control and experimental samples enables evaluation of these technical biases and need for extra quality filtering  transformation to account for the semiquantitative nature of HTS. Use of qualitative binary data (presence/absence) is not recommended, because of lower fit due to loss of the (semi)quantitative information and artificial equalization of potentially artefactual low‐abundance (including tag‐switch artefacts) and real high‐abundance OTUs . These files enable users to perform all steps of bioinformatics analyses including generation of OTU table and identification. This is important from several aspects such as confirming earlier findings with updated filtering procedures, addressing additional questions and performing metastudies using standardized filtering procedures. It is, however, discouraged to submit representative sequences of HTS‐derived OTUs to public databases because of their short length, potentially artefactual nature and unreliable taxonomic annotation. These environmental sequences would increase the proportion of poorly annotated and erroneous data and complicate identification in subsequent studies.www.datadryad.org) and DataOne (www.dataone.org). Darwin Core (https://rs.tdwg.org/dwc/) is the main standard for biodiversity data, which is linked to MIxS (https://gensc.org/mixs/) and MIMARKS (https://wiki.gensc.org/index.php?title=MIMARKS) standards by the Genomics Standards Consortium that are relevant to all gene sequence data and marker gene sequence data, respectively. Metagenomic data should follow MIxS and MIMS (https://wiki.gensc.org/index.php?title=MIGS/MIMS) standards (ten Hoopen et al., Curated OTU‐by‐sample matrices including technical and environmental metadata, representative sequences as well as taxonomic and functional annotations should be deposited in machine‐readable FAIR data format in specific data repositories such as Dryad Digital Repository (In publications, it is important to refer to any additional data in the supplement or data repositories. It is also important to record and describe precisely all analytical steps including specific options in data filtering, because this information provides important details about the data quality and stringency of filtering to the readers. Nilsson et al.  provide 7Only a fraction of available high‐throughput identification potential has been currently used in plant pathology. This is related to the practical surveillance‐oriented work of plant pathologists and entomologists but focus on human and animal subjects by molecular pathologists. Governmental plant health surveillance organizations need to follow certified protocols for diagnosis, which develop slowly due to time‐consuming tests. Limited budgets also hinder the possibility of purchasing high‐throughput analysis equipment by governmental institutions. Considering analysis costs and time, practicing pathologists would certainly take advantage of qPCR/ddPCR for real‐time quantification of specific pathogens and custom microarrays for simultaneous detection and quantification of multiple selected pathogens. In the nearest future, it may be possible to detect multiple organisms including pathogens using high‐throughput sequencing on portable pocket‐size sequencers as demonstrated for viruses using the Oxford Nanopore MinION platform (Loman et al., Other high‐throughput identification methods are more time‐consuming but also more sensitive and thus better suited for research purposes. Metagenomic and metatranscriptomic methods offer great potential when targeting viruses (Zhang et al., Because of great intraspecific resolution, high‐throughput fingerprinting and population genomics approaches offer enormous potential for diagnosis of aggressive strains or pathotypes and uncover their patterns of dispersal (O'Hanlon et al., For correct identification, community‐curated and taxonomically annotated reference databases are urgently needed. Such databases are maintained only for a few most important pathogen groups and cover the main barcoding marker genes (Park et al., Taken together, high‐throughput identification techniques offer great promise for detection and rapid identification of new pathogens and diseases in humans as well as tree and crop plantations and early warning systems such as the sentinel nurseries and botanical gardens. HTS has already demonstrated its usefulness in studies of soil‐ and plant‐associated microbial communities for detection of new potential pathogens and potentially invasive species before their introduction to the new environment and contact with new hosts. We predict that rapid monitoring methods such as nanopore sequencing, microarrays and nanotechnological biosensors will become particularly useful for early disease diagnostics and smart application of countermeasures such as biocides and biocontrol agents.M.C., L.T. and R.D. developed the concept; S.A. constructed the figure and contributed specific details together with C.M.‐R.; and L.T. wrote the article with input from co‐authors.