Bangladesh is one of the most densely populated nations that nonetheless has largely achieved staple self-sufficiency. This development has been enabled in part by the rapid proliferation of small-scale irrigation pumps that enabled double rice cropping, as well as by a competitive market system in which farmers purchase water at affordable fee-for-service prices from private irrigation pump owners. Excess groundwater abstraction in areas of high shallow tube-well density and increased fuel costs for pumping have however called into question the sustainability of Bangladesh’s groundwater irrigation economy. Cost-saving agronomic methods are called for, alongside aligned policies, markets, and farmers’ incentives. The study assesses different institutions and water-pricing methods for irrigation services that have emerged in Bangladesh, each of which varies in their incentive structure for water conservation, and the level of economic risk involved for farmers and service providers. Using primary data collected from 139 irrigation service providers and 556 client-farmers, we empirically examine the structure of irrigation service types and associated market and institutional dimensions. Our findings demonstrate that competition among pump owners, social capital and personal relationships, and economic and agronomic risk perceptions of both pump owners and farmers significantly influence the structure of irrigation services and water pricing methods. Greater competition among pump owners increases the likelihood of pay-per-hour services and reduces the likelihood of crop harvest sharing arrangements. Based on these findings, we explore policy implications for enhancing irrigation services and irrigation sustainability in Bangladesh. According to After independence in 1971, the Bangladesh government started investing heavily in irrigation, and until 1979, ground and surface water pumping was largely government managed. Original emphasis was on pumping surface and ground water, with the Bangladesh Agricultural Development Corporation (BADC) supplying surface water irrigation pumps, establishing deep tube wells (DTWs) and subsidized fuel to farmer groups and individuals . Small-sIrrigation services and pricing can vary substantially across and within locales . Bangladboro’ rice, with farmers applying 500–1000 l of water per kg of grain produced (Bangladesh is primarily a downstream delta – located in the eastern lower Indo-Gangetic Plains (IGP), a relatively fertile plain area that is intensively cultivated. Declining groundwater tables are particularly prominent in the drier northwestern IGP , and areproduced . Despiteproduced . Given t2Aspiring to achieve food self-sufficiency, the government of Bangladesh initially heavily invested in agricultural mechanization . During In the early 1980s, Bangladesh undertook market liberalization policies . EarlierIn 1982-83, Bangladesh relied on a mix of STWs, LLPs and DTWs to irrigate 1.52 million hectares of land . The totWith the expansion of groundwater abstraction, different forms of pump ownership and management have emerged. Different forms of irrigation water pricing and payment methods have subsequently emerged. Following the decentralization of BADC’s control over tube wells and LLPs, the primary payment method for irrigation water that emerged was the sharing one-fourth of the crop harvested by farmers with pump owners . Payment33.1p) and client-farmers (f) can be represented as:We hypothesize that irrigation water markets consisting of a pump owner and farmer-clients settle on an irrigation water payment method that tends to maximize the profit of the pump owner, while also minimizing farmers’ costs for purchasing irrigation services. These actors’ ability to achieve such a mutually agreeable equilibrium payment method is likely to be associated with the negotiation power of the pump owner and client-farmers, in addition to other environmental, market and exogenous factors. In a functional form, a mutually agreeable irrigation water payment method Several factors can influence the negotiation power of pump owners In contrast, a risk-taking pump owner might prefer to choose crop-sharing as his or her preferred method, from which they can more reliably claim a portion of grain produced at the end of the season. The degree of human and social capital, and specifically the relationship between pump owners and client-farmers, can also influence negotiation power, and as such the choice of the irrigation payment system. In functional form, this relationship can be described for farmers in Eq. We further hypothesize that the strength of the social relationship between pump owner and client-farmer can vary and is likely to have a considerable degree of influence on the payment method. For example, both pump owner and the client-farmer may be familial relatives, and they could, for example, can reside in the same village, shop in the same markets, and may pray in the mosque. These social interactions and their resulting relationships, therefore, can also influence irrigation water pricing payment methods and fees. As it is infeasible to estimate all three Eqs. For client-farmers:For pump owners:3.2We operationalize the conceptual framework to identify the personal and social relation factors as well as human capital endowment and environment domain that can affect the mode of irrigation water payment method. The resulting empirical model is specified as follows:iEV) that include:(1)the number of irrigation service providers (pump owners) operating in a sampled village;(2)a dummy variable that assumes a value of 1 if the community voluntarily participated in irrigation system maintenance, including within-field canal and/or field drainage operations (zero otherwise);(3)boro rice season (zero otherwise), and;a dummy variable  if there was no reported shortage of water in the pump command area at the peak of the (4)a dummy if the command area has a poor water drainage system  if excessive water accumulates within the irrigation scheme that causes stagnant waterlogging and that can potentially reduce crop productivity (0 otherwise).Explanatory variables include a vector of irrigation scheme specific variables familial relative dummies that assume a value of one if the pump owner and client-farmer have at least one blood relative employed in a government sector or who works as or around local politicians (zero otherwise);(6)iHC.At the time of data collection, we asked the sampled respondents on how much risk they usually take in their daily economic activities and asked them to score it in between zero to ten, in which with zero indicating a completely risk-averse attitude of the sampled pump owners and farmer-clients, and 10 indicating a strong preference for risk-taking. The self-assessed general risk scores provided by pump owners and farmer-clients are included in The vector of variables iHC includes some additional variables for the pump owner and client-farmer:(7)number of years of schooling;(8)the household size measured by the number of immediate family members;(9)boro season; andarea total land cultivated land (ha) during the (10)dummy variables that assume a value of one if the major occupation of the pump owner and farmer-client involves off-farm employment (zero otherwise).The vector isocial relation index includes the social interaction indicators between a pump owner and client-farmer. This index is constructed by applying the Principal Component Analysis (PCA) to dummy variables for pump owner and client-farmer familiar relations, residence the same village, and for owners and clients who pray in the same mosque or temple. These variables are intended to capture the influence of the social and personal relationships on irrigation water payment method. Details of the process of generating the relation index are included in Northern region dummy is a dummy variable that assumes a value of one (zero otherwise) if the surveyed village was located outside greater Barishal region that is characterized by a higher potential for and use of surface water irrigation  in the completed 2013–14 season.This study is based on primary data collected from April 22 to June 8 in 2015 from 139 pump owners who provided irrigation services, and from 556 farmer-clients who purchased their irrigation services. In Bangladesh, dry season or maize . As the We focused on two prevailing smallholder irrigation systems: surface water irrigation using LLPs that are common in Barishal division, and ground-water extraction using STWs, which are more common in north and west Hourly payment, in which farmer-clients pay cash per hour to pump owners (includes both with and without client fuel given our limited sample size),(2)Seasonal with client fuel, in which client-farmers pay a fixed cash rate per season per unit of land, and in addition, supply fuel or provide fuel costs for the full season,(3)Seasonal without client fuel, where farmer-clients pay a flat cash rate per season per unit of land without additional fuel charges, and(4)Crop sharing, also known as share cropping, in which farmer-clients trade an agreed share of harvested grain and/or straw  in lieu of cash for irrigation.We identified four major irrigation payment methods  includinAll of the sampled irrigation service providers were using the centrifugal pumps and practiced flood irrigation. No solar irrigation pumps were identified, and none of the sampled farmer-clients or irrigation service providers used alternate wetting and drying (AWD) or System of Rice Intensification (SRI) techniques, which was widely promoted but also critiqued in Bangladesh and South Asia, to address the increasing water scarcity in rice-based cropping systems . Overallboro rice in 2013–14. Each sampled command areas had < 19 client-farmers with a mean command area of 5.74 ha  80 per hourmodality . The houmodality . Total sOn average, an irrigation engine consumed 401 l/ha of diesel equivalent fuel for an entire season for irrigation, however, the fuel consumption in crop-sharing arrangements was the lowest  and to a lesser extent by the pump owner (23%) in study areas . HoweverOn average, a sampled client-farmer had 4.6 years of formal schooling, 0.81 ha of land, and five family members. Seven percent were engaged in off-farm economic activities . By cont5We hypothesize the different irrigation services provide different incentive mechanisms to conserve water from farmer-clients’ point of view. Using the seasonal with client fuel service (a two-part tariff payment) as the default group in The number of irrigation service providers in a village is positively associated with the hourly service payment system for both pump owner and client-farmer, whereas the crop sharing method is strongly and negatively associated. The findings suggest that competition among irrigation service providers thereby encourages the adoption of relatively water conserving irrigation services. Voluntary participation in irrigation command area maintenance, which we used as a proxy for community relationship, is negatively associated with seasonal payment without farmer-client fuel supply systems, in addition to crop sharing irrigation services for both pump owner and client-farmer. This suggests that the presence of a strong community relationship discourages flat rate irrigation services that are less efficient directly in terms of fuel – and indirectly water.boro rice farming riskier and less productive, with crop sharing allowing the partitioning of risks between farmers and pump owners while reducing the cash costs for the latter.The availability of irrigation water during the season was positively associated with crop sharing in the case of pump owners and all types of irrigation services (compared to two-part tariff methods) for client-farmers. The poor drainage dummy was positively associated with crop sharing for both the pump owner and client-farmer, reflecting problems with canal and field water management in lower-elevation areas of Barisal division . Poor flHaving a blood relative in a government job or active in politics positively affected only the seasonal flat rate without fuel use by farmers. This could potentially be indicative of the farmers’ increased negotiation power. Where feasible, more risk-taking farmers likely prefer to reduce their reliance on seasonal flat rate without fuel, and crop sharing systems, which provide forms of risk sharing e.g., , and areFinally, the social relation index between pump owner and farmers has negatively associated the likelihood of crop sharing, suggesting that social bondage can increase incentives to opt for socially desirable payment systems, but without such capital, less risky and potentially more cost-effective irrigation services may prevail. Compared to the two-part tariff payment as a base, the hourly, seasonal flat rate and crop sharing payment systems are less prevalent in northern or western Bangladesh, where civil infrastructure is relatively more developed with greater cropping intensity .6Bangladesh’s privatization policies and the liberalization of machinery imports contributed to the rapid proliferation of small-scale mechanized pumps and irrigation services provision. Using primary information collected from 556 farmers and 139 pump owners, this study demonstrates that different irrigation service payment systems have emerged in Bangladesh over time, with a shift away from crop sharing towards cash-based options. This study unpacks the development of varying types of pricing systems and explores the factors that contributed to their differentiation.boro rice land during the 2013–14 winter season. This equates to roughly 1.23 billion liters of diesel being consumed for in a single season. Stationary burning of one liter of diesel results in the release of 2.69 kg of direct and embodied carbon dioxide (CO2)  . In a siOur study demonstrates that Bangladesh’s irrigation payment systems are mainly groundwater-based, with traditional centrifugal pumps used for water abstraction. Recently, projects led by the International Maize and Wheat Improvement Center (CIMMYT) have introduced axial flow pumps (AFP) that are suitable for surface water irrigation and respond to governmental policy priorities championing increased surface water use . AFPs caThe study confirms that geography and pump type can influence the irrigation service choice, in which farmer-clients and pump owners in relatively riskier environments, such as Barisal division are more likely to choose crop sharing. Social capital and risk profiles also substantially influence irrigation payment types. Public support reducing crop production risks could potentially encourage risk-averting farmers to choose more socially desirable water conserving irrigation services, such as hourly or two-part tariff methods that both constitute a variable production cost and, hence, provide an incentive to reduce the quantity of water applied. Finally, our findings indicate that competition among pump owners could contribute to the emergence of socially desirable irrigation services with an in-built incentive for water conservation. These hypotheses, however, need to be verified with further research. A major policy recommendation, therefore, will be how to encourage and maintain healthy competition among service providers to enhance smallholder adoption of sustainable and environmentally sounds irrigation practices alongside increased food security given Bangladesh’s pursuit of the Sustainable Development Goals.


Considering the great energy and biomass demand for cell survival, cancer cells exhibit unique metabolic signatures compared to normal cells. Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent neoplasms worldwide. Recent findings have shown that environmental challenges, as well as intrinsic metabolic manipulations, could modulate HNSCC experimentally and serve as clinic prognostic indicators, suggesting that a better understanding of dynamic metabolic changes during HNSCC development could be of great benefit for developing adjuvant anti-cancer schemes other than conventional therapies. However, the following questions are still poorly understood: (i) how does metabolic reprogramming occur during HNSCC development? (ii) how does the tumorous milieu contribute to HNSCC tumourigenesis? and (iii) at the molecular level, how do various metabolic cues interact with each other to control the oncogenicity and therapeutic sensitivity of HNSCC? In this review article, the regulatory roles of different metabolic pathways in HNSCC and its microenvironment in controlling the malignancy are therefore discussed in the hope of providing a systemic overview regarding what we knew and how cancer metabolism could be translated for the development of anti-cancer therapeutic reagents. Malignancies of the head and neck influence a variety of anatomic sites, including the oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, and salivary glands . The oncAlthough cancer is generally considered to be a genetic disease , inconsiIn early years, HNSCC-specific metabolic fingerprints were defined mainly by immunohistochemistry staining analysis and serological examination. For example, cellular retinoic acid binding protein (CRABP) expression was enriched in tumor tissues compared with its adjacent normal tissues , while aFrom a broad point of view, the correlation between systemic diseases and HNSCC cancer is an interesting topic worthy of further investigations. Taking Diabetes mellitus (DM) as an example, although for more than fifty years clinicians have reported that DM and cancer are diagnosed within the same individual more frequently, both diseases are complex with multiple subtypes, making it difficult to conclude the association between them . PreviouAs discussed in recent studies, by using NMR spectroscopy, HNSCC-specific metabolic features correlated with an acquired EGFR-TKI resistance ; the intHyperactive glycolytic metabolism is the most promising metabolic change in highly proliferating cells . DespiteThe progression of biogenetic changes during cancer development, based on Warburg’s theory, is: insufficient respiration acts early during malignant transformation, followed by a compensatory energy production through glycolysis to continuously ferment lactate in the presence of oxygen, leading to deficient oxidative phosphorylation (OxPhos) that is irreversible ,44. IndeROS, a by-product of ETC, are molecules that could capture a singlet oxygen atom in mitochondria under a normal physiological condition. Under a certain pathological status, an accumulating ROS generation results in an imbalanced respiration chain, thereby leading to oxidative stress, a well-known toxic physiological stimulus for various diseases including cancer formation ,67. ProvIt is widely known that the TCA cycle acts as an essential metabolic pathway for the cell energy supply. The numbers of compounds were therefore developed to modulate the TCA cycle aiming to regulate cellular bioenergetics-mediated phenotypes in cancers. For HNSCCs, it was reported that Lanthanide could inhibit HNSCC growth in glucose free conditions by suppressing cytosolic Malic enzyme 1 (ME1) that converts malate into pyruvate, leading to a decreased pyruvate level and consequent lactate production, accompanied by increased mitochondrial areas. This study suggested a metabolic shift away from aerobic glycolysis toward oxidative phosphorylation in response to lanthanide treatment . A more At a genetic basis, mitochondria possess their own transcriptional codes. Mitochondrial DNA (mtDNA) is located in the mitochondrial matrix and encodes 37 subunits of the tETC complex involved in the OxPhos system. Based on previous findings, HNSCC cells utilize aerobic glycolysis preferentially over the mitochondrial metabolism, suggesting that a decreased OxPhos activity is a potential predisposition for HNSCC development ,81. IndeCancer cell proliferation also requires the duplication of cellular macromolecular components during each cell division, making lipid metabolism a principal physiological pathway for maintaining cell architecture, growth and physiological homeostasis ,87,88,89As for HNSCC, the significance of the lipid metabolism in controlling the pathological transformation, in comparison with glycolysis- and mitochondria-related cues, is much less emphasized. It was firstly studied from the findings that cholesterol-lowering drugs, including lovastatin and simvastatin, exhibited a great anti-HNSCC effect by facilitating cell apoptosis and attenuating 1 integrin mediated cell migration, implying that circulating lipids might play a role in regulating HNSCC malignancy ,104. FurLike glucose, there are major differences in the utilization and metabolism of amino acids in tumors when compared to normal cells. All twenty amino acids could serve differently as important regulatory cues to many of the physiological processes, including the biosynthesis of macromolecules, cellular redox homeostasis, post-translational and epigenetic modifications and anaplerostic reactions ,118,119.In addition to intrinsic amino acids, cells could obtain amino acids externally via different transporters, including the L-type amino acid transporter 1 (LAT1), which is specific for the branched-chain and aromatic amino acid intake; the system ASC amino-acid transporter-2 (ASCT2), which is responsible for the neutral amino acid uptake; and SLC7A11, also named xCT, an bidirectional exchanger for cysteine-glutamate. Previous studies have found that these transporters are enriched in many cancers and that their expression is correlated with a worse survival rate ,127,128.Including different amino acids in the discussion, individual amino acids play differential roles in regulating tumor cell malignancy. For example, methionine-derivative S-adenosyl methionine (SAM) is a cofactor acting mainly as a methyl donor to cytosine in DNA during epigenetic modification . A recenGlutaminolysis, a metabolic pathway for converting glutamine into glutamate via glutaminase, is widely documented as playing an essential role in regulating neoplastic phenotypes. Immunohistochemistry staining and Western blot analysis both showed a higher glutaminase expression in metastatic HNSCC tissues than in normal tissues. Furthermore, shRNA-mediated silencing for glutaminase abrogated the HNSCC proliferation and downregulated the cellular glutamate level. At the cellular level, a positive correlation between glutaminase and aldehyde dehydrogenase (ALDH) was reported, while the administration of glutamine could enhance the ALDH expression and restored the tumor growth in ALDHloCD44lo cells, suggesting that glutaminolysis-related manipulations could be a potential method for the development of a HNSCC stem cell based therapeutic regimen . AnotherTaken together, amino acid metabolic cues are important for HNSCCs, although much effort should be put in better identifying the significance of different amino acids in controlling the HNSCC physiology under the same experimental settings. In addition, based on the fact that different amino acid metabolisms crosstalk to each other, it becomes essential to apply isotope labeling methods to trace amino acid related metabolites, and their associations with phenotypic alterations and metabolic shifts. In this way, the metabolic plasticity of HNSCC cells could be more clearly illustrated, and key amino acid metabolic pathways could be uncovered.Although the majority of cancer research focuses on investigating the roles of the protein-coding genes in controlling oncogenicity, the exon-coding strands only consist of 3% of the whole human genome . The regMost ncRNA-mediated metabolic controls for tumorigenesis rely on regulatory cues derived by the surrounding environment. Cancer progression is highly dependent on the interactions between the tumor cell and the tumor microenvironment (TME); TME is comprised of various cell types, such as endothelial cells, cancer-associated fibroblasts (CAF), and immune cells, which may cross-talk with cancer cells via cytokines, growth factors, proteases or hormones . It therIn HNSCC, the upregulation of LncCAF (FLJ22447) in cancer-associated fibroblasts (CAFs) upregulates cytokine IL-33, supports tumor growth and leads to a poor prognosis . Using tIn addition to LncRNA, several studies supported the roles of miRNAs in orchestrating cross-talks between tumor cells and tumor stroma . In HNSCAside from the LncRNAs and miRNAs detected in the tumor or its microenvironment, the analysis for serological and salivary LncRNAs and miRNAs as potential diagnostic tools has recently been carried out ,171,172.Tumor cells rely on external nutrients for proliferation and survival, and it is therefore not surprising that the cancer milieu plays a crucial role in controlling tumourigenesis ,187,188.The first observation for determining the importance of CAFs in HNSCC was described by Rosenthal et al., showing that elevated TGF 1 was detected in the stromal counterpart of HNSCCs compared to normal mucosa ; the strIn theory, the immune system could protect cells from attacks by microorganisms and pathological cells including tumor cells, in order to maintain homeostasis in the body. Although TILs could be found in clinic HNSCC tumor tissues, the elevated expression of the death-associated domains Fas and Fas ligand (FasL) was also detected , indicatHypoxia stimulates a complex oncogenic alteration in tumors . Oxygen Cellular metabolism is highly dynamic, and the regulation between environmental and intracellular metabolic cues is closely intertwined. In order to translate the current knowledge about cancer metabolism for the development of clinical therapeutic schemes, several points should be addressed. First, the effects of using combinational therapy of conventional chemo/radiotherapy plus metabolic modulators in treating HNSCCs were rarely examined. While working mechanisms vary for conventional therapies (mainly targeting cell growth) and metabolism-mediated anti-cancer agents, it would be interesting to determine whether combinational therapies could be superior to single treatments. Second, in order to manipulate tumorous metabolic cues in a broader manner, an ideal platform for screening potential reagents that suppress cancerous metabolic molecules and simultaneously upregulate less active metabolic factors in cancer cells would be required. Last, it is likely that HNSCC tumors and stromal cells exhibit reciprocal metabolic cues that could be important in helping cancer cells escape from immune attacks and, at the same time, in exhausting immune cells in TME. It therefore becomes essential to define metabolic molecules that display opposite expression pattern in tumors/CAFs and tumor-associated immune cells on a single-cell population level. In this way, the candidate compound(s) could be more efficiently suppressing the malignancy and stimulating immune cell activity .


In recent years, high-throughput next-generation sequencing technology has allowed a rapid increase in diagnostic capacity and precision through different bioinformatics processing algorithms, tools, and pipelines. The identification, annotation, and classification of sequence variants within different target regions are now considered a gold standard in clinical genetic diagnosis. However, this procedure lacks the ability to link regulatory events such as differential splicing to diseases. RNA-seq is necessary in clinical routine in order to interpret and detect among others splicing events and splicing variants, as it would increase the diagnostic rate by up to 10–35%. The transcriptome has a very dynamic nature, varying according to tissue type, cellular conditions, and environmental factors that may affect regulatory events such as splicing and the expression of genes or their isoforms. RNA-seq offers a robust technical analysis of this complexity, but it requires a profound knowledge of computational/statistical tools that may need to be adjusted depending on the disease under study. In this article we will cover RNA-seq analyses best practices applied to clinical routine, bioinformatics procedures, and present challenges of this approach. In recent years, the use of next-generation sequencing (NGS) for the diagnosis of Mendelian or rare genetic disorders has entered routine clinical practice. The increasing ability to sequence entire genomes in a cost-effective manner has allowed the identification of approximately 260 novel rare genetic diseases per year . Focusintrans-acting factors (proteins and ribonucleoproteins) and cis-acting elements (pre-mRNA sequences), including exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE), and intronic splicing silencer (ISS) elements  can help to shed light on the possible pathogenicity of variants of unknown significance (VUS) identified through DNA sequencing studies such as WES and WGS, as it provides direct insight into the transcriptional alterations caused by VUS and thus improves diagnostic rates . Alternaelements , that excis-acting regulatory elements that are located either in core consensus sequences  or in regulatory elements that modulate spliceosome recruitment . Degradation of aberrant transcripts by a nonsense-mediated decay (NMD) mechanism results in ASE of the SPAST wild-type allele , cofunded by European Regional Development Fund (ERDF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the hippocampus plays a critical role in spatial and episodic memories, the mechanisms underlying memory formation, stabilization, and recall for adaptive behavior remain relatively unknown. During exploration, within single cycles of the ongoing theta rhythm that dominates hippocampal local field potentials, place cells form precisely ordered sequences of activity. These neural sequences result from the integration of both external inputs conveying sensory-motor information, and intrinsic network dynamics possibly related to memory processes. Their endogenous replay during subsequent sleep is critical for memory consolidation. The present review discusses possible mechanisms and functions of hippocampal theta sequences during exploration. We present several lines of evidence suggesting that these neural sequences play a key role in information processing and support the formation of initial memory traces, and discuss potential functional distinctions between neural sequences emerging during theta vs. awake sharp-wave ripples. The ability of brain circuits to generate precise sequences of neuronal activity may underlie certain forms of sensory-motor processing and learning  which provide membrane resonance and subthreshold oscillations at theta frequency  and cholecystokinin (CCK) basket cells, as well as axo-axonic cells, is minimal; and different classes of GABAergic neurons have different preferred firing phases . Independent initiation of phase precession in successive cells would automatically yield theta sequences  preferentially occurs on the descending phase of theta. It is thought to arise from CA3 and to propagate along the Schaffer collaterals to the stratum radiatum. Medium gamma oscillations  tend to occur near the theta peak and may emerge from MEC inputs targeting the stratum lacunosum-moleculare  arising at the theta peak decreased, while gammaS power (30–45 Hz) arising at the theta trough increased toward the end of the fixation period. The authors hypothesized that CA1 received predominant inputs from the EC at fixation onset, providing information about ongoing context. This signal would then elicit auto-associative intra-hippocampal processing in CA3 to recall the required action, or to look up in memory the appropriate task sequence given the ongoing context. This scenario, reminiscent of the model of Chance . These results suggest that alternating gammaS and gammaM states allow the hippocampus to switch between prospective and retrospective modes, possibly to prevent interference between memory retrieval and encoding.In the first study, Bieri et al.  found thM than during theta cycles dominated by gammaS , but not both , compared to when the mice used a place strategy, when spikes emitted during gammaS and gammaM periods contributed equally to the hippocampal representation. A possible interpretation is that during sequence strategy trials, the hippocampal network retrieves sequences of cell assemblies stored in CA3, while during place strategy trials, hippocampal activity would be equally driven by the recall of memory stored in CA3 and by landmark-related sensory information conveyed by the EC.The spatial strategy of the animal may also bias the balance between gammal et al.  trained As hinted previously, theta sequences do not appear to be entirely driven by sensori-motor signals. For instance, Gupta et al.  reportedConvincing and direct evidence that cognitive mechanisms can regulate theta sequences was provided by Johnson et al. , who recBecause the mechanisms underlying theta sequences remain poorly understood and may involve many interacting factors , interfering with theta sequences is challenging. To the best of our knowledge, only three studies have reported task performance deficits linked to a degradation of theta sequences.status epilepticus, a condition that leads to mesial temporal lobe epilepsy (MTLE). These rats had altered phase precession and temporal organization of firing among pairs of neurons, along with deficits in spatial learning in the Morris water maze. This suggests that memory impairments stemmed from impaired temporal coding, consistent with a role for theta sequences in normal memory function.In the first study, Lenck-Santini et al.  investigIn the second study, Robbe and Buzsáki  examinedWang et al.  also repIn summary, the above studies are consistent with the view that theta sequences are involved in the formation and recall of spatial memory, possibly by enabling the synaptic changes that underlie the storage of initial memory traces . Our previous work has established that passive travel alters phase precession in most place cells , but from the occurrence of dynamic network patterns with a well-documented function, namely trajectory replay underlying memory consolidation. As a consequence, this alleviates the ambiguity discussed earlier, that behavioral impairments could result either from memory deficits, or from impairments in planning and decision making.bona fide trajectories until theta sequences progressively emerge between P17 and P32.Interestingly, additional evidence for a link between theta sequences and sleep replay was recently provided by Muessig et al. , who recFinally, over the last 15 years, an ever growing number of studies have addressed the network substrates of memory using causal approaches. However, these studies have generally failed to consider the temporal dimension of neuronal activity, such as manifested in cell assemblies and sequences. Experimental exploration of engrams Semon,  has beneper se. This would potentially restore the temporal patterns obliterated by the synchronous experimental stimulation. A parallel can be made with fRMI studies showing robust and consistent activation patterns in response to similar stimuli, despite the low temporal and spatial resolution of the technique  of slow wave sleep  of planned trajectories in working memory. During VTE, extended neuronal sweeps may support deliberative processes. Awake replay could also support trajectory planning, on-line consolidation and reconsolidation, as well as on-line retrieval of long-term memory. Thus, it remains currently unclear which specific functions are sustained by theta sequences, awake replay, or both.Finally, a growing number of studies have provided evidence that hippocampal activity can also convey non-spatial information, including about objects, odors, sound sequences, time, goals, fear, etc. (e.g., Wood et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The number of druggable tumor-specific molecular aberrations has grown substantially in the past decade, with a significant survival benefit obtained from biomarker matching therapies in several cancer types. Molecular pathology has therefore become fundamental not only to inform on tumor diagnosis and prognosis but also to drive therapeutic decisions in daily practice. The introduction of next-generation sequencing technologies and the rising number of large-scale tumor molecular profiling programs across institutions worldwide have revolutionized the field of precision oncology. As comprehensive genomic analyses become increasingly available in both clinical and research settings, healthcare professionals are faced with the complex tasks of result interpretation and translation. This review summarizes the current and upcoming approaches to implement precision cancer medicine, highlighting the challenges and potential solutions to facilitate the interpretation and to maximize the clinical utility of molecular profiling results. We describe novel molecular characterization strategies beyond tumor DNA sequencing, such as transcriptomics, immunophenotyping, epigenetic profiling, and single-cell analyses. We also review current and potential applications of liquid biopsies to evaluate blood-based biomarkers, such as circulating tumor cells and circulating nucleic acids. Last, lessons learned from the existing limitations of genotype-derived therapies provide insights into ways to expand precision medicine beyond genomics. In the past decade, the field of oncology has witnessed substantial changes in the way patients with cancer are managed, with departure from a “one-size-fits-all” approach and increasing focus on precision medicine based on genomic variants. Cancer precision medicine is defined as “the use of therapeutics that are expected to confer benefit to a subset of patients whose cancer displays specific molecular or cellular features (most commonly genomic changes and changes in gene or protein expression patterns)” . In certFirst and foremost, the important interactions between research and standard of care testing in precision cancer medicine must be highlighted. Large-scale research studies may identify new biomarkers with clinical utility, which can then be taken up as a new standard of care clinical diagnostic test to maximize the benefit to the patient population. Many tumor-specific molecular alterations, including protein overexpression, mutations in driver genes, or rearrangements, are well-proven predictive biomarkers of response to selective targeted therapies, with additional biomarkers rapidly emerging. Clinical molecular pathology analysis has therefore become an indispensable laboratory tool that can be used to characterize tumor biology and to drive therapeutic decisions.EML4-ALK translocation in non-small cell lung cancer (NSCLC) and IDH1/2 mutations in glioma) [.Conventional tests such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are fundamental precision medicine tools in daily practice , with ma glioma) –7. The u glioma) , 8–10. F glioma) , 12.As the number of druggable gene aberrations and predictive biomarkers grow in oncology, NGS technologies have increasingly substituted for conventional techniques, such as single-gene testing, and for targeted mutational platforms in routine molecular pathology. Conventional techniques have high sensitivity and specificity in detecting actionable mutations with proven benefit from matched targeted therapies or in identifying mutations that are associated with resistance to specific drugs –16. Howe. The number of genes included in these panels can vary, ranging from 20–30 to over 400–500 genes. Although the clinical utility of assessing all of the genes that are included in large panels is currently uncertain, the benefit of simultaneous multi-gene testing using NGS and the low incremental cost of including additional genes are motivators for using more comprehensive genomic profiling in the clinic.NGS can be limited to a pre-specified group of genes (targeted gene panels), can focus on the coding regions of all of the base pairs of the genome (whole exome sequencing (WES)), or can involve the analysis of the entire tumor genome, including the intronic regions (whole genome sequencing (WGS)). The choice between these approaches depends on several factors, including the final application of tumor testing , the results required, technical efficiency, and cost  end up being treated with genotype-matched drugs –28. MultEGFR and ALK genes, with panel testing offering additional opportunities for targeted therapy to fewer than 5% of patients [EGFR T790M mutation confers resistance to first-generation EGFR tyrosine kinase inhibitors (TKI) in NSCLC) or to suggest potential synergistic drug combinations  [EGFR-mutant NSCLC (such as osimertinib) [BRAF-mutant NSCLC [A recent retrospective analysis in NSCLC showed no survival gain in patients who underwent genomic profiling using multi-gene targeted panels compared to patients who underwent only single-gene testing of patients . Howeverelanoma) , 33. Theertinib) , 35 and nt NSCLC , 37 and nt NSCLC , which hAdvances in sequencing technologies such as WGS have facilitated the simultaneous detection of single nucleotide variants (SNV), CNV, and structural rearrangements such as gene fusions, leading to greater diagnostic yield of actionable findings in tumor samples. The value of performing comprehensive genomic profiling is exhibited by a recent study that characterized 2520 samples of metastatic tumors with paired normal tissue. WGS was used to identify the catalog of genetic mutations found in each metastasis, thus illuminating the genomic changes present in the metastases of 22 solid tumors, and 62% were found to contain at least 1 actionable event . SimilarThe quality, quantity, and availability of tumor tissue from cancer patients pose challenges to the clinical implementation of precision medicine. The processing of formalin-fixed, paraffin-embedded fragments can alter nucleic acids, and low tumor content in tumor samples can decrease test sensitivity or lead to false-positive mutation calls . In addiAn emerging field that may ameliorate some tumor sample issues is the testing of circulating tumor-specific markers. These include circulating tumor cells (CTC) or circulating tumor DNA (ctDNA), as well as RNAs, proteins, or metabolites, that are present in body fluids such as the blood, urine, and peritoneal or cerebrospinal fluid –54. LiquPotential clinical uses of blood-based CTC and ctDNA that are not offered by tumor tissue sequencing include monitoring the persistence of radiologically undetectable tumors , prediction of recurrence  , monitorEGFR mutations in patients with NSCLC and the methylation of the SEPT9 promoter as screening for colorectal cancer [. Topics that remain to be addressed include the high variability of CTC and plasma ctDNA levels between individual patients that result from inconsistent shedding and the impact of disease burden and/or cancer type on circulating nucleic acids [To date, two tests based on plasma ctDNA analysis have been FDA-approved for clinical use: the testing of l cancer , 67, 70.ic acids .Beyond CTC and ctDNA, other circulating tumor biomarkers such as RNA, proteins, and metabolites are still in early phases of development and need to be explored properly. At present, there are no FDA-approved assays for the detection and analysis of these biomarkers and their clinical utility remains unclear . CirculaNational Center for Biotechnology Information (NCBI) ClinVar, Online Mendelian Inheritance in Man (OMIM)) that compile previously reported germline polymorphisms may also aid in differentiating tumor-specific somatic variants from non-pathogenic DNA variations in patients for whom no available matched normal tissue or blood is available [The interpretation of the clinical impact of tumor variants in the context of a specific cancer and for individual patients is an active field of study in precision cancer medicine . To facivailable , 83.. Other global harmonization initiatives for variant curation and interpretation, such as the Global Alliance for Genomics Health (GA4GH) Variant Interpretation for Cancer Consortium (VICC), have also been proposed [One of the major downsides of having multiple knowledgebases is the dispersion of the genomic information. New user-friendly tools have been developed to integrate the knowledge from established databases on diseases, genes, variants, and drugs  in a single space, with the ultimate goal of improving clinical interpretation by healthcare professionals . In addiproposed .The implementation of guidelines and consensus to standardize somatic variant annotation, classification, and reporting is critical to enable the interpretation of variants across institutions and professionals. The Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists have published a series of recommendations for the classification and reporting of somatic variants in cancer patients, which are based on their clinical significance and the available supporting evidence . EvidencBRAF), and assessment of microsatellite instability proved to have greater sensitivity than IHC in identifying Lynch syndrome in patients with colorectal cancer [BRCA1 and BRCA2 for PARP inhibitor therapy may reveal inherited germline mutations in these genes. To address these issues, recent guidance from the European Society of Medical Oncology Precision Medicine Working Group recommends that germline-focused analysis should be performed during tumor-only genomic profiling to identify variants with high allele frequencies (> 20–30%) and selected genes of clinical relevance [The detection of clinically relevant germline mutations in patients undergoing tumor genomic profiling has been reported , 87. Thil cancer . For ovaLarge-scale genomic sequencing is currently available through academic institutions and private enterprises and is now funded in some jurisdictions but not in others; for instance, funding is now provided by Medicare in the USA. Target–drug matching can become increasingly complex as more information becomes available through the use of large panel tests or WES/WGS approaches. There are expanding numbers of patients with complex genomic data that are in need of interpretation. In order to exploit the potential of NGS-driven therapy fully, a formal entity such as a molecular tumor board (MTB) should exist that brings interdisciplinary expertise into the evaluation of patients who have advanced cancer to indicate when alteration-driven treatment is advisable. These multi-disciplinary teams typically include oncologists, research scientists, bioinformaticians, pathologists, medical geneticists, genetic counselors, and genomicists, among others. They examine each patient’s clinical, pathologic, and molecular information, review the literature and available resources, carry out discussions to reach a consensus if possible, and make treatment suggestions . PreviouPublished studies have identified a knowledge gap and lack of confidence of physicians in their ability to interpret sequencing data. For instance, 22% of physicians at a tertiary cancer center reported a lack of confidence in their genomic knowledge, so there is clearly a need to educate oncologists in interpreting genomics data . YoungerThere are various challenges to implementing a successful MTB; for instance, it is not always possible for members to meet in person, the MTB may not always be accessible by community oncologists, and there is a lack of standard quality requirements and guidelines on how to run an MTB and make treatment decisions . A solutThe application of NGS may provide the treating physician with a list of druggable alterations. However, approved drugs are often inaccessible to biomarker-positive patients who have different tumor types because of a lack of reimbursement for drugs that are being used beyond their labeled indications. As a result, patients either need to be treated within the auspice of clinical trials or enrolled in compassionate access programs. Most clinical trials only cover a minority of potential genomic treatment indications and often have strict inclusion and exclusion criteria. Some molecular screening programs have performed gene panels to identify patients for opportunistic enrollment into early phase trials of targeted agents, whereas others have channeled patients to prospective biomarker-driven studies that sought out specific aberrations , 96–98.BRAF mutations are found across multiple tumor types) [. As a result, the paradigm of precision oncology has shifted to “pan-cancer” biomarker-based approaches for therapeutic selection. The predictive value of NTRK fusions as biomarkers of response to TRK kinase inhibitors  is a successful illustration of this approach. Both drugs have now been approved by the FDA to treat all solid tumors carrying NTRK fusions, and thus, TRK kinase inhibitors represent the second approved group of tissue-agnostic drugs in cancer, following pembrolizumab for patients with MSI-high tumors [NTRK fusions, a diagnostic algorithm based on the incidence by tumor type and NTRK expression by IHC testing has been proposed as a more efficient detection strategy in routine practice [Large-scale tumor profiling studies using NGS have revealed significant genomic similarities, with shared actionable alterations in driver genes, among different tumor types  , 100. Ash tumors . Neverthpractice –105.BRCA1 and BRCA2 responded to carboplatin, whereas those with a BRCA mutational signature and no germline variant did not respond [As discussed, genomic profiling for cancer precision medicine has a significant focus on finding discreet driver mutations that are associated with therapeutic targets or that are of diagnostic or prognostic value. An additional genomic tool in cancer are genomic “profiles” that harbor similar patterns of gene expression or of inherited or somatic mutations across multiple genes or genomic regions. With proper analysis, it is possible to group patients into subcategories for response, outcomes, or other clinical features. Mutational signatures expand genomics beyond the simple focus of discreet variant detection, with risk profiles reported in numerous cancer types including hepatocellular carcinoma, breast cancer, brain cancer, and diffuse large B cell lymphoma –108. The respond . More cl. Numerous other breast cancer recurrence risk testing platforms that use expression signatures are also available and incorporated into clinical practice guidelines [The most advanced use of gene signatures is gene expression profiling from RNA sequencing (RNAseq), gene expression microarrays, or other single-molecule enumeration methods that are used to subclassify tumors into gene expression signatures. For example, gene expression arrays are used to provide consensus molecular subtyping of colorectal cancer . Mutatedidelines . These sidelines . The Woridelines . In thisidelines . A similidelines .DNMT and EZH2, genome-wide epigenetic maps of DNA methylation and histone modifications are being developed  [IDH2 and SRSF2 genes promote leukemogenesis through coordinated effects on the epigenome and RNA splicing [BRAF mutations or KRAS mutations, respectively [Epigenetic changes modify the genome in order to modulate transcriptional activity that ultimately generates a permissive or restrictive architecture for cell growth and proliferation . The episortium) , 121. Thsortium) . Simultasplicing . Genome-splicing . High anectively . AlthougPTEN loss, or mutations in JAK or other IFNγ-related genes) can be informative of resistance to immune checkpoint inhibitors [Beyond the protein expression of immune checkpoint molecules such as PD-L1, genomic analyses also play a role in predicting response or resistance to IO agents . Tumor mhibitors , 136–138hibitors .. Current pitfalls and potential solutions are discussed below.The field of precision oncology is moving from isolated genomic analyses towards a multi-omic approach to achieve a better understanding of tumor biology and to increase treatment opportunities. The ACNS02B3 brain tumor biology study, led by the Children’s Oncology Group across several institutions, represents a successful example of expanding molecular profiling beyond genomics. In this study, five distinct tumor molecular subgroups were identified on the basis of IHC, genomics, epigenetics, and transcriptomic analyses, which were reproducible in patient-derived xenograft models and thus allowed for in vivo drug sensitivity tests . Beyond BRCA1/2 are associated with tumorigenesis in only a few types of cancer, namely breast, ovarian, prostate, and pancreatic cancers, and that there is little benefit in treating other cancer types that harbor such mutations with PARP inhibitors [Tremendous progress has been made in the field of precision medicine, with ever-increasing numbers of patients being tested and new biomarkers being developed leading to expanded therapeutic opportunities, but challenges remain. The results from target–drug matching initiatives have been disappointing to date, as most of these have matching rates of only 5–10% and the objective responses in genotype-matched patients have been modest . The education of patients prior to testing is essential, but how best to execute this is unknown . A helpfTraditional clinical trial designs may not offer an efficient investigation of precision medicine, and as a result, more flexible trial designs have been developed. Adaptive studies include inbuilt opportunities to modify one or more specified trial elements on the basis of intermediate data analysis. For instance, treatment arms may be opened or closed on the basis of provisional findings at pre-specified points, such as emerging evidence of response to treatment. This can increase efficiency by facilitating the selection of the dose, sparing patients from being exposed to ineffective doses, and reducing cost and duration of clinical development . The evoERRB2/HER2 amplification [FGFR alterations [PIK3CA [EGFR mutation or ALK translocation after completion of standard therapy [A number of basket trials around the world are currently recruiting patients, some of which are highlighted in Table fication , FGFR alerations , or PIK3 [PIK3CA  mutation therapy .Table 4The Drug Rediscovery Protocol (DRUP) is an ongoing Dutch adaptive precision oncology trial that facilitates the use of approved drugs beyond their approved indication in rare cancer subgroups. Initial results from the two subgroups that have completed data accrual have been published. The first was in agnostic microsatellite instable (MSI) tumors treated with nivolumab. The European Medicines Agency has not yet approved the use of checkpoint inhibitors in this setting, but on the basis of the positive results from DRUP, the Dutch regulatory agency has now approved the use of these drugs in this indication. The second cohort in microsatellite stable colorectal cancer with TMB between 11 and 22 mutations per megabase showed limited clinical benefit, leading to the closure of the arm , 172.By and large, current platform trials are exploring targeted agents given as monotherapies. Genomic alterations do not always lead to oncogenic pathway activation or addiction, whereby certain tumor cells become dependent on a single activated oncogenic protein or pathway. As a result, targeting of multiple driver and/or resistance pathways using combinatorial approaches may be required for optimal antitumor activity . The iPRhttps://uhnddp.ca) [Numerous components are involved in automating the matching of patients to clinical trials, including creating a database and then establishing a method to search the database to match patients to trials. In addition, the maintenance of up-to-date comprehensive databases is necessary for the automation of patient–trial matching . The Phanddp.ca) . Furthernddp.ca) . The usenddp.ca) .The implementation of precision medicine through molecular profiling technologies has increasingly been integrated with standard clinicopathological evaluations to enhance diagnosis, prognostication, and prediction of clinical outcomes. Although there have been clear successes in the era of molecular characterization, the utility of NGS and other omics-based tests remains unproven on many fronts. A vision for the future of precision medicine will integrate comprehensive multi-omic tumor characterization, dynamic monitoring of liquid biopsy samples, annotation that is automated through advancements in artificial intelligence but guided by experts’ clinical input, the enrollment of patients into innovative clinical trials that not only test molecular profile–drug matching but also investigate the utility of different drug-assignment algorithms , and theAdditional file 1. Advantages and disadvantages of NGS-based approaches.


Radiotherapy (XRT) improves local control and plays an essential part in the reduction of extensive surgical procedures in the management of patients with soft tissue sarcoma (STS). Historically, surgery is performed first followed, on indication, by adjuvant XRT (AXRT), balancing the gain in local control to toxicity. A landmark study by Rosenberg et al. demonstrated no statistically significant difference in local recurrence (LR), disease-free survival (DFS), and overall survival (OS) between STS patients after amputation and patients with limb-sparing surgery and AXRT.A statistically significant reduction of the risk of LR of more than 50% by AXRT was demonstrated in this study.4Prospective studies are needed to evaluate further the impact of XRT on clinical outcome in STS patients. Concerning neoadjuvant XRT, with the sarcoma mass still in situ, clinical trials should be designed with appropriate translational side studies in order to distinguish the more radiation sensitive subtypes. Obviously, STS represents many different tumours, each with their own clinical behaviour. Currently, all are irradiated to a uniform schedule.


Instead of original abutments, compatible abutments are often selected for financial reasons. The present study aimed to evaluate mechanical outcomes, microleakage, and marginal accuracy at the implant-abutment interface of original versus nonoriginal implant abutments. Search strategy encompassed literature from 1967 up to March 2017 to identify relevant studies meeting the inclusion criteria. The following electronic databases were consulted: PubMed database of the U.S. National Library of Medicine, Embase (Excerpta Medica dataBASE), and the Grey Literature Database (New York Academy of Medicine Grey Literature Report). Quality assessment of the full-text articles selected was performed. Abutments were classified in original (produced by the same implant manufacturer), nonoriginal certified , and nonoriginal compatible (produced by a third-party milling center for similar connections). A total of 16 articles fulfilled inclusion criteria and quality assessment and were selected for the qualitative analysis. All of the included studies were in vitro research with high or moderate risk of bias and reported data from 653 implant abutments. Original and nonoriginal certified abutments showed better results in terms of mechanical outcomes, microleakage, and marginal accuracy compared to nonoriginal compatible abutments. Following the clear warnings coming from the present systematic review, clinical suggestions regarding the effect of a nonoriginal abutment can be drawn. However, in vivo, long-term, randomized controlled trials are needed to provide definitive clinical conclusion about the long-term clinical outcomes of original and nonoriginal abutments. Current commercially available dental implants are mostly two-part systems: the implant is positioned at the bone level and, once osseointegrated, loaded using an abutment supporting the prosthetic restoration. Microgaps at the implant-abutment interface (IAI) are unavoidable and are the consequences of the micro-tolerances between components required during the industrial manufacturing.The implant connection has not been demonstrated to prevent bacterial contamination , 2. In cThe majority of the marginal bone loss (MBL) associated with the biologic width reestablishment has been reported during the first year after implant placement, while hereafter, in patients with adequate levels of oral hygiene, the marginal bone levels stabilize over years. However, micromovements of the implant-abutment complex  represent the second key factor for the determination of MBL and biologic width . A recenIn daily practice, practitioners and dental technicians often select compatible abutments for financial reasons. The differences in design are possibly related to patent issues that do not allow for exact replication of components and/or related to the precision level and the quality control of materials used during the manufacturing process. For the latter, nonoriginal components may differ in the design of connecting surfaces, shape, dimensions, and material and have shown higher leakage values , 12. TheThe aim of the present systematic review was to evaluate mechanical outcomes, microleakage, and marginal accuracy at the implant-abutment interface of original versus nonoriginal implant abutments.  P: samples of implant-supported single crowns;  I: original stock or computer-assisted design and computer-assisted manufacturing (CAD/CAM) abutments;  C: nonoriginal abutments (certified or compatible);  O: mechanical outcomes , microleakage (microgap), and marginal accuracy (fit).  S: in vitro studies.The present systematic review was written according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The focOriginal abutment is an abutment produced by the same implant manufacturer based on original nominal dimensions. These could be stock or CAD/CAM. Nonoriginal abutment is an abutment produced by a different implant manufacturer (or third-party company). These could be certified or compatible. Nonoriginal certified abutment is an abutment produced by a third-party milling center, directly or indirectly certified for certain implant companies. These could be gold cast or CAD/CAM. Nonoriginal compatible abutment is an abutment produced by an implant company compatible with other implant systems with the same implant-abutment interface. It might also be manufactured by a generic producer not directly involved in the implant manufacturing. These could be stock or CAD/CAM.  Original and nonoriginal abutments were defined as follows:∗” OR “Dental Implant Abutment∗”) AND .An initial search strategy encompassing the English literature from 1967 up to February 2018 was performed online to identify relevant studies that met the inclusion criteria. The following electronic databases were consulted: PubMed database of the U.S. National Library of Medicine and Embase (Excerpta Medica dataBASE). According to the AMSTAR (A MeaSurement Tool to Assess Systematic Reviews) checklist, the Grey Literature Database was screened in the New York Academy of Medicine Grey Literature Report in order to find possible unpublished works. Screening was performed by an expert examiner (MT). A second reviewer (LC) reassessed the included and excluded studies. The electronic databases were searched using a combination of Boolean keywords, MeSH, and several free-text terms. The Boolean search algorithm employed to find potentially relevant literature was developed by extracting the keywords of relevant literature found on preliminary scoping searches and included the following terms:  .The following outcome measures were analyzed: (1) mechanical outcomes ; (2) microleakage (microgap at the implant-abutment interface); (3) marginal accuracy (fit). The same reviewers assessed the quality of the searched manuscript and the risk of bias in the included studies according to a modification of the guidelines provided by the CONSORT statement for the evaluation of randomized controlled trials .A total of 175 potentially relevant titles and abstracts were found after the electronic search. During the first stage of selection, 154 articles were excluded based on the titles and abstracts. In the second phase, complete full-text articles of the remaining 21 publications were evaluated and 11 articles were excluded since they did not fulfill the inclusion criteria. Six publications were added from manual extraction of the reference lists of selected relevant articles. Finally, a total of 16 articles that fulfilled inclusion criteria and quality assessment required for the present systematic review, reporting data from 653 implant abutments, were selected in the qualitative analysis. A diagram of the search strategy is shown in The selected studies were published between 2003 and 2017. All the included studies were in vitro research reporting data on single-implant-supported restorations with different implant-abutment interface. Overall, 188 abutments were original: ten studies reported data from original stock abutments (n=108) and seven studies reported data from original CAD/CAM abutments (n=80). Conversely, 465 abutments were nonoriginal: seven studies reported data from nonoriginal, compatible, stock abutments (n=237); five studies reported data from nonoriginal, compatible, CAD/CAM abutments (n=73); and six studies reported data from nonoriginal, certified, CAD/CAM abutments (n=155) .According to a modified CONSORT checklist of items for reporting in vitro studies of dental material, two out of 16 studies were at moderate risk of bias , 18, whiMechanical outcomes has been defined as mechanical deformation, damage, and failure under applied forces. Gigandet et al.  demonstrFour studies , 27, 28 When testing the maximum load capacity, Kim et al.  reportedLeuter et al.  demonstrOn the contrary, Yilmaz et al. , conductMicroleakage may be defined as the clinically undetectable passage of bacteria, fluids, molecules, or ions between a cavity wall and the restorative materials. Microleakage directly depends on the marginal accuracy of the components . Alonso-Pérez et al.  evaluateConversely, in a publication on the analysis of the vertical marginal fit after static and dynamic load, after thermocycling with artificial saliva, the authors found that original abutments were highly superior to nonoriginal certified abutments . Da CunhNone of the included studies reported data from radiographic marginal bone loss, as well as biological or mechanical complications due to the nature of the selected study design.The aim of the present systematic review of in vitro studies was to evaluate any difference in mechanical outcomes, microleakage, and marginal accuracy between original and nonoriginal abutments in implant-supported restorations. In fact, dental implant abutments can be either original or nonoriginal. Original abutments are manufactured by the same implant company. Recently, due to the wide range of customized abutments request, international milling centers proposed “certified” nonoriginal connections. At the same time, original designs have been incorporated in a various number of competing companies that offer the so-called “compatible” connections, and their relative nonoriginal, compatible, dental implant abutments. None of the included studies reported data from radiographic marginal bone loss, as well as biological or mechanical complications.To the best of our knowledge, at the time of writing this systematic review, there were no in vitro studies comparing mechanical outcomes and marginal accuracy at the implant-abutment interface of original versus nonoriginal implant abutments. Furthermore, with so many variables of implant types, connections, materials, methods of manufacture of the restorations existing, it is not possible to combine data from multiple studies.The majority of the included studies , 22, 28 Although reported exponential source of bias  makes the translation to the clinical practice difficult, clinical relevance of this review could be extrapolated. In fact, dimensional tolerances express the possible space between two connecting components and the permissible limits of variation in a physical dimension deviating from a nominal dimension as noted in this review. The tolerances between implant and abutment may play a major role in an implant-abutment connection, defining its mechanical outcomes. Although high quality manufacturing cannot prevent tolerances, data collected in the present systematic review might suggest that differences between original and nonoriginal certified or noncertified abutments might be defined by the size of implant/abutment tolerances.In two-piece implant, a microgap is created at the implant-abutment interface. Previous systematic review and meta-analysis have shown that oral microbiome can proliferate in this microgap and affect peri-implant tissues, causing inflammation and peri-implant diseases . PreventThe results of this review also demonstrate that CAD/CAM abutments provide comparable, if not better, clinical outcomes when compared, in vitro, with conventional abutments. However, existing evidence is weak. Schepke et al., in an in vivo randomized controlled trial comparing the use of a CAD/CAM customized zirconia abutments with stock zirconia abutments, for the rehabilitation of a single tooth replacement, failed to find any improvement in clinical performance or patients' satisfaction .The main limitation of the present systematic review was the presence of only in vitro studies reporting insufficient methodological details. Therefore, due to the in vitro nature of these studies and the high risk of bias, the outcomes reported in these systematic reviews should be carefully interpreted. In fact, the in vitro values do not represent the clinical situation because only vertical load application can be used for the comparative analysis, and the numerous biological parameters that influence mechanical outcomes in vivo are not taken into account , 35. LonAdditional limitation might be represented by the fact that none of the included studies reported data from multiple implants, not allowing a complete generalization of the study outcomes.In the light of this preclinical outcomes, long-term consequences of nonoriginal abutments should be analyzed to evaluate the clinical relevance of their mechanical instability. For this purpose, long-term randomized controlled trials aimed to compare original to nonoriginal restorations and the microbiological contamination at the connection level could answer this clinical question.Lower incidence of mechanical failure and higher marginal accuracy were reported for originals compared to nonoriginal abutments. Differences between nonoriginal certified and nonoriginal compatible were highlighted. However, clinical conclusions regarding the effect of a nonoriginal abutment cannot be drawn. In fact, data were derived from “in vitro” studies with a high risk of bias.Therefore, there is an urgent need for well-designed randomized clinical trials to provide more information about the long-term clinical outcomes of original and nonoriginal abutments.


As our understanding of cancer cell biology progresses, it has become clear that tumors are a heterogenous mixture of different cell populations, some of which contain so called “cancer stem cells” (CSCs). Hallmarks of CSCs include self-renewing capability, tumor-initiating capacity and chemoresistance. The extracellular matrix (ECM), a major structural component of the tumor microenvironment, is a highly dynamic structure and increasing evidence suggests that ECM proteins establish a physical and biochemical niche for CSCs. In cancer, abnormal ECM dynamics occur due to disrupted balance between ECM synthesis and secretion and altered expression of matrix-remodeling enzymes. Tumor-derived ECM is biochemically distinct in its composition and is stiffer compared to normal ECM. In this review, we will provide a brief overview of how different components of the ECM modulate CSC properties then discuss how physical, mechanical, and biochemical cues from the ECM drive cancer stemness. Given the fact that current CSC targeting therapies face many challenges, a better understanding of CSC-ECM interactions will be crucial to identify more effective therapeutic strategies to eliminate CSCs. The extracellular matrix (ECM) is a major structural component of the tumor microenvironment and comprised of a network of biochemically distinct components, including fibrous proteins, glycoproteins, proteoglycans, and polysaccharides. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or modified . IncreasCollagens constitute the main structural element of the ECM and are the most copious type of fibrous proteins within the interstitial ECM. Collagens play a role in tissue development by providing mechanical strength, altering cell adhesion, promoting cell migration . StudiesGlycoproteins, which make the ECM a cohesive network of molecules by linking cells together with structural components, include fibulin, fibrillin, laminin, fibronectin, vitronectin, tenascin-C, Secreted Protein Acidic and Rich in Cysteine (SPARC), periostin (POSTN), thrombospondin, mucins (MUCs) and nidogen . CSCs ovProteoglycans are glycosylated proteins composed of a core protein and one or several covalently attached sulfated glycosaminoglycan chains and are present in the ECM of connective tissues. Proteoglycans play a crucial role in ECM assembly and cell signaling. They bind to growth factors, cytokines and other ECM molecules and act as co-receptors to assist ligand and cell surface binding to modulate downstream signaling. Several proteoglycans  are highly expressed by CSCs and their roles in cancer stemness are summarized in Polysaccharides, a chain of monosaccharide repeats linked through glycosidic bonds, fill the interstitial space and buffer physical stress on the ECM. Hyaluronic acid  is a high-molecular-mass polysaccharide that constitutes a major component of interstitial gels, especially in soft connective tissues. In tumors, HA is produced by both tumor stroma and tumor cells, and its binding to the cellular receptor CD44 activates intracellular signaling  to promote cell survival, cancer stemness, motility and invasion by cytoskeletal reorganization. High levels of HA are produced by CSCs and HA–CD44 interaction has been shown to promote acquisition of CSC characteristics and chemoresistance in breast, ovarian and head and neck CSCs .Physical properties of the ECM such as rigidity, porosity and topography impact various anchorage-dependent CSC functions. The interstitial ECM, mainly composed of collagens, proteoglycans and hyaluronan, provides a physical barrier that hinders the transport of solutes, water and chemotherapeutic drugs. In this regard, it has been shown that cisplatin, a chemotherapeutic drug frequently used to treat various solid tumors, extensively binds to collagen fibers in tumors . BindingTumor ECM is typically stiffer than normal tissue ECM due to overexpression of many ECM components  and ECM-modifying enzymes  . MechaniThe ECM can provide external cues that induce EMT, one of the cellular transformation processes that has been shown to route some cancer cell types from a differentiated to a stem cell state . CollageThe ECM also promotes CSC self-renewal. In this regard, collagen I has been shown to preserve stemness in malignant and non-malignant stem cells by activating transcriptional programs that induce self-renewal . BindingThe ECM might serve as a reservoir for factors that aid in the sustenance of CSCs. Embryonic stem cells (ESCs) have been shown to utilize matrix metalloproteases 1 (MMP1) to release ciliary neurotropic factor (CNTF) from an ESC-derived matrix, which enhances ESC self-renewal though JAK/STAT3 signaling , a pathwThe ECM serves as a functional repository for a plethora of factors that dynamically modulate the tumor microenvironment to promote CSC metabolism. Focal adhesion formations transduce ECM signaling into the tumor cells and activate the PI3K pathway which increases glycolysis, in addition to activating glutamine signaling in a Ras- and Myc- dependent manner. Furthermore, a stiff ECM acts as a driver of glycolysis in CSCs . On the in vitro and tumor initiating potential in vivo (in vitro and in vivo (Solid tumors frequently contain highly hypoxic regions and tumor hypoxia is positively associated with poor prognosis. Hypoxic tumor cells express stem cell markers, are highly undifferentiated and exhibit enhanced clonogenic potential  in vivo . Further in vivo . In addi in vivo . Abrogat in vivo . ECM rem in vivo . Since aExtracellular matrix can profoundly influence recruitment of immune cells into the tumor microenvironment. CSCs can evade immune surveillance by altering this microenvironment to favor their survival. For example, ECM drives the activation of pro-survival pathways such as PI3K/AKT, which has been shown to facilitate immune evasion in CSCs . ECM proCurrently, there are several inhibitors targeting various aspects of ECM-induced cancer stemness that are undergoing clinical testing. For example, the CD47 blocking protein TTI-621  is curreAlthough the above drugs may effectively reduce the number of CSCs, there are still many potential challenges that ECM components in a tumor microenvironment may set that could interfere with an otherwise successful treatment regimen. Firstly, ECM proteins have been shown to act as a physical barrier, making drug delivery to cancer cells more difficult. Secondly, ECM proteins can de-differentiate non-CSCs into CSCs, which makes eliminating all CSCs more challenging. Thirdly, ECM plays a role in modulating immune cell recruitment, hence, potential immunotherapeutic strategies could be hindered by dysregulated ECM components. Finally, the ECM has a very complex and dynamic nature: different ECM molecules are expressed in a time and tissue-specific manner where various isoforms of the same molecule can play opposing functions in cancer stemness in a context-dependent manner. Considering these concerns, it is crucial that future studies further elucidate the role of ECM components on cancer stemness in order to design therapies that effectively eradicate all CSCs.All authors conceptualized the content and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.