Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy. Second, application of nanobody technology in investigating protein–protein interactions is reviewed, with emphasis on possible uses in mass spectrometry. Finally, we discuss the potential value of nanobodies in studying protein function, and we focus on their recently reported application in targeted protein degradation. Throughout the review, we highlight state-of-the-art engineering strategies that could expand nanobody versatility and we suggest future applications of the technology in the selected areas of fundamental research. Camelidae. These antibodies are devoid of the light chain and lack the first constant domain. Consequently, the antigen-binding fragment of HcAbs is solely composed of a single variable domain, referred to as VHH (variable domain of the heavy chain of HcAbs), single-domain antibody or nanobody, which is only ~15 kDa in size. The variable domains of conventional IgGs and HcAbs comprise three complementarity-determining regions (CDRs) that constitute the paratope of the antibody  of conventional antibodies, resulting in similar binding affinities. In dromedary nanobodies, these long loops are often connected by a disulfide bond that restricts their flexibility and consequently, favors antigen binding. Normally, FR2 region of VHs contains highly conserved hydrophobic amino acids participating in the interaction with the VL (variable domain of the light chain). As this region is water-exposed in nanobodies, the hydrophobic amino acids are substituted by hydrophilic residues, which reduce the likelihood for aggregation. This explains the high solubility of nanobodies (Since the discovery of heavy chain only antibodies (HcAbs) in 1993 by the Hamers-Casterman’s group    or explom (T3SS) , 17. SecIn this review, we assess the current status of nanobodies as research tools in diverse facets of fundamental research . Moreover, we focus on the adaptability of nanobodies, or how engineering can expand their versatility, and we discuss future opportunities given the current know-how. As the use of nanobodies in diagnostics and therapy does not fall within the scope of this paper, we refer the reader to some excellent recent reviews , 19.Several studies confirmed the usefulness of nanobodies as equivalent detection surrogates for antibodies in immunocytochemistry Table . de BruiN-hydroxysuccinimide ester-labeling , STORM , or PALM  , 35. TheN-hydroxysuccinimide ester-labeling (see later in this section)  –24. The section) . They lasection) . Accordisection) . GFP andsection) .Using GFP or RFP nanobodies as detection tool has some advantages. First, high-affinity GFP and RFP nanobodies are commercially available , which mN-hydroxysuccinimide (NHS) ester-labeling 1 of primary amines 2 (R-NH2) is the most widespread labeling strategy  derived from  in mice .11 was incorporated into thymosin β4 peptide. Photooxygenation of the furan moiety results in the formation of a 4-oxo-enal moiety 12. Subsequent addition of a NH2NH-coupled label, transforms the furan-containing peptides into pyrrolidinone-based fluorescent probes 13  or nanobodies equipped with an epitope tag that allows antibody detection Table . These iSeveral studies report the use of a GFP-targeting nanobody to study protein–protein interactions Figure ; Table 2Künzl and coworkers studied vacuolar sorting in plants using a GFP nanobody sensor . SolubleGFP nanobody has also been exploited as targeting moiety for upconversion nanoparticles (UCNPs) in lanthanide-based resonance energy transfer (LRET) imaging. In simple terms, lanthanide-doped UCNPs are able to convert two or more near-infrared photons into one UV/Vis photon. In its turn, this photon can sensitize a neighboring acceptor fluorophore. The UCNPs were functionalized with anti-GFP nanobody to target a bait EGFP-fusion protein. On the other hand, prey protein was fused to an acceptor fluorophore. Sensitized fluorescence upon LRET from the UCNPs can only be detected when bait and prey interact (in)directly. As proof-of-concept, the indirect interaction between mitochondrial outer membrane (MOM) proteins Tom20 and Tom7 was successfully visualized using UCNP LRET .In the latter three studies, a high-affinity GFP nanobody was used to study protein–protein interactions in living cells, albeit combined with different techniques . The nanobody was utilized as delocalization tool, thereby enriching bait and eventually prey at defined subcellular locations  , 44, or bona fide interaction partners. Combining this strategy with Virotrap, a lysis-free protein interaction analysis method, could improve the study of protein complexes, as lysis-sensitive protein complexes are preserved. Virotrap implies trapping a bait protein, together with its putative interaction partners, inside protective virus-like particles (VLPs) that bud from cells. Following antibody-based enrichment and lysis of the VLPs, protein complexes can be analyzed by Western blot or MS. Packing of bait in VLPs is achieved by expression of p55 HIV-1 GAG-bait fusion protein   . Similarraction) . CombiniIn contrast to mitochondria and endoplasmic reticulum, peroxisomes are endowed with the ability to import oligomeric protein complexes –59. CombAs will be discussed in the next section, the nanobody-binding epitope could be used to identify “weak” spots in proteins, which offers opportunities for small molecule development. Hydrogen/deuterium exchange MS (HDX-MS) allows fast epitope characterization with small amounts of sample and has already frequently been used to characterize antibody epitopes –52. In b2-adrenergic receptor (β2AR)  with the purpose of knocking out (one or more) protein function(s), causing measurable effects Table . The ultr (β2AR) –71. Thesormation . Inhibitormation .Existing (inhibitory and non-inhibitory) nanobodies can be engineered to expand their usefulness as a tool for investigating protein function Table . EquippiNon-invasive intrabodies can also be engineered in such way that they can interfere with normal cell biology. As such, Jullien and colleagues transformed their H2A-H2B histon chromatibody into an invasive tool by coupling the nanobody to an E3 ubiquitin ligase . ExpressOur understanding of protein function has improved considerably by technologies that manipulate protein levels, such as RNAi or Morpholino antisense oligonucleotides. However, as these methods operate upstream of the protein level, they depend on the turnover rate of their target, thus resulting in limited depletion of long-lived proteins. Moreover, they frequently generate off-target effects , 79. To via its C-terminal domain, and with a substrate adaptor protein via its N-terminus. The substrate adaptor protein mediates substrate specificity and recognizes its target directly  or indirectly , the latter necessitating an additional adaptor protein  for target binding. The CRL complexes ubiquitylate proteins and as such, mark them for degradation by the proteasome  and even across nanobodies from different camelid species. Presence of the cognate antigen confers nanobody stability, whereas antigen absence results in proteasomal degradation of the nanobody and its associated tags. As such, it is possible to endow antigen-expressing subsets of cells with particular features. Tang and colleagues for instance exclusively labeled ACH-2 HIV-1 positive T-cells for flow cytometry , and soluble high-affinity targeting moieties that can easily be produced. Moreover, it is possible to engineer nanobodies in such a way that they display a desired function or set of functions , without interfering with its binding characteristics. Hence, they are highly adaptable. These favorable characteristics stimulated their use as research tools in diverse aspects of fundamental research. Undoubtedly, in future years, new applications will continue to surface.EB and JG wrote the manuscript. All authors reviewed 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. The reviewer, GH, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Hepatitis C virus (HCV) morbidity appears to be high in Mongolia. Yet, the scale and nature of the infection burden is not well-understood. Our study’s objective is to systematically review and synthetize all available epidemiological data on HCV antibody (Ab) prevalence, ribonucleic acid (RNA) prevalence, incidence, risk factors to HCV exposure, and circulating HCV genotypes/subtypes among different at-risk populations. Additionally, we aim to estimate national population-level HCV-Ab prevalence and the number of HCV chronically infected individuals in the population of Mongolia.p value ≤0.05). In order to identify relevant reports, we will search PubMed, Embase, and Index Medicus for the Southeast Asian region. Additionally, we will search Mongolian scientific and medical journals not indexed in PubMed or Embase and the archives of Mongolian local conferences. Lastly, the literature search will be supplemented by checking references of the included reports and identified reviews. We will use broad search criteria with no language or time restrictions. Meta-analyses will estimate pooled HCV-Ab prevalence , and pooled RNA prevalence among HCV-Ab positive individuals in the general population. Age-adjustment of estimates will be conducted.Our systematic review will be reported based on the items outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2009) statement. All reports with primary data collected from surveillance or observational studies on Mongolian populations will be eligible for inclusion if the study sample size is greater than 25. Included reports need to present studies that use biological assay for HCV-Ab ascertainment. We will consider three primary outcomes of interest: HCV-Ab incidence, HCV-Ab prevalence, and HCV genotypes/subtypes among different at-risk populations. In addition, two secondary outcomes of interests will be also collected: HCV RNA prevalence, and unadjusted and/or adjusted statistically significant risk factors for HCV exposure  contains supplementary material, which is available to authorized users. Mongolia is a lower-middle-income country located in Central Asia and bordered by Russia and China . Thirty-Hepatitis C virus (HCV) morbidity appears to be high in Mongolia. Yet, the scale and nature of the infection burden is not well-understood. The objective of this protocol is to describe the methodology developed for a systematic review and synthesis of all available epidemiological data on HCV antibody (Ab) incidence, HCV-Ab prevalence, ribonucleic acid (RNA) prevalence, risk factors to HCV exposure, and circulating HCV genotypes/subtypes among different at-risk populations. Additionally, we aim to estimate national population-level HCV-Ab prevalence and the number of HCV chronically infected individuals among the general population of Mongolia.Our systematic review will be reported based on the items outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2009) statement . The curAll reports with primary data collected from surveillance, cross-sectional, cohort, or case-control studies will be eligible for inclusion if the study sample size is greater than 25. Case reports, case series, reviews, qualitative studies, editorials, commentaries, letters to editors, author replies, and animal studies will be excluded.As in earlier reviews –16, the Included reports will be those including data on Mongolian populations in Mongolia. Therefore, studies conducted on Mongolian nationals outside of Mongolia will be excluded.Included reports need to present studies that used biological assays for HCV-Ab ascertainment. Reports will be considered ineligible if the studies are based on self-report, or classification of HCV as non-A non-B hepatitis.p) value ≤0.05).We consider three primary outcomes of interest: HCV Ab-incidence, HCV-Ab prevalence, and HCV genotypes/subtypes among different at-risk populations. In addition, two secondary outcomes of interest will be also collected: HCV RNA prevalence and unadjusted and/or adjusted statistically significant risk factors for HCV exposure  assessment at study-level, studies will be classified based on an adaptation of the Cochrane principles . This adRegarding the ROB assessment at outcome-level , an HCV-Extracted outcome measures  will be reported according to the study population’s risk of acquiring HCV infection (likelihood of being exposed to HCV: (1) populations at low-risk: these include populations such as the general population, blood donors, pregnant women, and healthy adults and children. (2) Populations at intermediate-risk: these include populations such as healthcare workers, medical students, patients with sexually transmitted diseases, HIV infected persons where the main suspected mode of transmission is sexual, female sex workers, men who have sex with men, homeless people. In addition, as mobile men in Mongolia are considered to be at a greater risk for HIV and STIs due to a greater probability of having casual sex partners and/or having sex with female and/or male sex workers , mobile Regarding the quantitative synthesis, we will pool primary measures including HCV Ab-incidence, HCV-Ab prevalence, and HCV genotypes/subtypes among different at-risk populations, and secondary outcomes as relevant such as RNA prevalence. Outcome measures with sample sizes of at least 25 participants will be eligible to be included in the meta-analyses. Outcome measures estimated from stratifications by subpopulation, and/or sex, and/or study period, and/or region, and/or age, will be prioritized for inclusion from each given included report. As such, if a report provides regional and national outcome measures for the same population, we will include the regional measures. Binomial method  will be All meta-analyses will be conducted using random-effects models, to account for expected heterogeneity in outcome measures across studies. Outcome measures will be weighed by the inverse-variance of the double-arcsine transformed proportions , accordiQ test will be conducted [Q test p value of <0.10 will be considered as significant. I2 heterogeneity measure and its 95% CI will be calculated to assess the magnitude of between-study variation that is due to heterogeneity across studies rather than chance [In order to assess heterogeneity in outcome measures across studies, forest plots will be inspected visually and Cochran’s onducted . A two-sn chance . The 95%n chance , 30.Q test for subgroup differences [Qbetween subgroups test p value of <0.05 will be considered as significant.In order to explore the variation in pooled HCV-Ab prevalence and heterogeneity in risk of exposure between subgroups, we will conduct subgroup meta-analyses and meta-regressions as warranted by the extracted data. We will pool HCV-Ab prevalence by at-risk subpopulation , time period, study location or region, sex, and age group. Comparisons between subgroups will be conducted using the ferences . Qbetwet test p value is <0.10.We will investigate sources of heterogeneity between studies using meta-regression . PotentiAll data analyses will be conducted in R v.3.1.1.   using thF test with a significance threshold at 0.01.Pooled age-specific HCV-Ab prevalence trend among the general population will be assessed using a polynomial regression model. Multiple R-square will be estimated to evaluate how well the model fits the pooled age-specific HCV-Ab prevalence trend. Analysis of variance will be conducted to identify which order of the polynomial regression model should be selected. The significance of the observed trend will be assessed by the Using the de facto population distribution by age group provided by the Census 2010 as extracted from the database of the Population Division of the United Nations Department of Economic and Social Affairs  and usinodds proportion defined as prevalence/. In order to test the asymmetry in the funnel plots, we will conduct Peter’s tests [Regarding publication bias assessment, we will use funnel plots to explore small-study effect on the pooled estimates. A minimum of ten studies will be required for this assessment. We will produce funnel plots of log (odds proportion) against study size , with od’s tests .As in an earlier systematic review , we willThis systematic review and meta-analysis will determine HCV-Ab prevalence and its distribution, RNA prevalence, HCV-Ab incidence and HCV genotypes/subtypes in Mongolia among different at-risk populations. The methodology to be used in the proposed study will ensure a robust synthesis of available data. The ultimate aim of our work is to provide empirical evidence necessary for researchers, policy-makers, and public health stakeholders to set research, policy, and programming priorities for HCV prevention, control, and eventually elimination in Mongolia.


Nanoporous gold (np-Au) has intriguing material properties that offer potential benefits for many applications due to its high specific surface area, well-characterized thiol-gold surface chemistry, high electrical conductivity, and reduced stiffness. The research on np-Au has taken place on various fronts, including advanced microfabrication and characterization techniques to probe unusual nanoscale properties and applications spanning from fuel cells to electrochemical sensors. Here, we provide a review of the recent advances in np-Au research, with special emphasis on microfabrication and characterization techniques. We conclude the paper with a brief outline of challenges to overcome in the study of nanoporous metals. There exists a large inventory of porous materials with a variety of pore sizes and morphologies to benefit numerous applications. The requirements of each application and basic scientific curiosity demand innovative processing and characterization techniques to understand the fundamental properties of these materials. A subset of porous materials is porous metals, which are of special interest due to their catalytic activity, electrical conductivity, and mechanical properties. Among porous metals, nanoporous gold (np-Au) has attracted distinct attention from the scientific community and industry alike. This surge of interest is evidenced by the recent increase in the number of papers published on np-Au: the ~25 papers appearing in the thirteen years spanning 1992–2005 has blossomed into more than 80 in the last four years (2006–2009). Controllable pore morphology of np-Au provides a highly adaptable system for the fundamental study of wide range of mechanical and surface properties . T. Tin sitmembrane . They elAn enabling technology for probing the mechanical properties of materials has been the fabrication of nano-pillars b. These Powerful sensor applications of np-Au will likely find use in handheld devices that can identify minute amounts of pollutants in water sources in remote settings or quantify disease-markers in blood for point-of-care applications. Such technologies will require successful integration of np-Au sensors with peripheral electronics or microfluidic channels, where the ability to produce np-Au sensor components with conventional microfabrication techniques is crucial. Photolithographic patterning techniques, employing photo-sensitive polymer coatings as stencil masks or etchant-resistant masks  with different properties  can be patterned on a single substrate. This allows for a highly parallel survey of alloy and final np-Au properties . We envision this technology to greatly facilitate optimization of np-Au properties for specific applications, most notably for sensors.The most frequently used method to modify morphology has been thermal treatment, which leads to coarsening of pores and ligaments ,57,58,59One method to prevent undesired coarsening of np-Au films is to prevent surface diffusion of gold. This has been effectively demonstrated with the addition of platinum to the initial alloy  or deposet al. suggested a relationship between halides in dealloying electrolyte and the final nanoporous morphology [0.35Ag0.65) with KI-containing electrolytes.Dursun rphology . They haThe mechanical constraints, and hence stress evolution in np-Au structures exposed to thermal treatment, result in profound differences in porosity evolution , as showet al. developed an interesting approach to create np-Au with multimodal pore size distribution, where they electroplated a secondary Au-Ag alloy  onto an existing np-Au template and subsequently dealloyed it to reveal a composite pore morphology [et al. compared the effects of thermal treatment and acid treatment in coarsening of pore and ligament sizes of np-Au sheets [i.e., HNO3 and HCl) result in remarkably different morphologies and the underlying mechanisms are unclear.Ding rphology . Hakamadu sheets . They obu sheets . It is iet al. suggested that the volume reduction may be a result of small scale plastic deformation due to homogeneous slip in small ligaments or by climb of lattice dislocations [A major problem in synthesis of np-Au films is the formation of voids and/or cracks in films, hindering their application as functional coatings. A significant proportion of the crack formation in np-Au films is due to volume contraction during dealloying. This phenomenon has been observed by different groups ,50,62. PShrinkage during dealloying becomes especially problematic in fabrication of constrained microstructures, such as doubly-clamped beams . Figure Similar crack/void formations are evident in blanket np-Au films on rigid substrates ,44, as wet al. observed a phenomenon where complete intergranular or transgranular fractures of Au-Ag wires and foils can be induced by applying a small tensile stress to structures during dealloying  [Other groups have emphasized the importance of microstructural length scale of np-Au and ligament size in ductile-brittle transitory behavior of np-Au, as well as its fracture strength ,58. Barnill wet) . It is pResearchers have utilized numerous existing methods and novel techniques for the analysis of alloys and np-Au structures. In this section, we provide a review of current techniques of characterization and summarize key findings.Residual stress in structures is of great importance, in blanket films and miniature structures alike. Compressive residual stress may lead to delamination of blanket films and buckling of freestanding structures. Conversely, tensile stress is the main culprit for cracking and for some delamination. 0.4Ag0.6 beams) also increased with thermal treatment temperature ; however, the residual stress was generally lower compared to blanket films, in part due to minimized substrate interaction with the beam, hence more freedom to relax. Lee at al. measured residual stress of microfabricated np-Au beams  synthesized from white gold leaves (Au0.37Ag0.63) and obtained ~65 MPa [et al. increases the yield stress of np-Au beams, thereby leading to higher residual stress values compared to the beams fabricated by “permanent buckling” approach. This is a striking example of how different experimental conditions can lead to large variations in residual stress in np-Au freestanding structures. Nevertheless, there is limited literature on residual stress of np-Au structures.Thermal annealing of Au-Ag blanket films on silicon wafers led to temperature-dependent increases in residual stress . This is attributed to work hardening of the alloy during thermal cycling-related compression-tension . Regardl ~65 MPa . It is d ~65 MPa ,43; howeet al. used two different nanoindenter probes  to determine elastic modulus (~11 GPa) of 0.5 mm-thick np-Au samples with ~46% porosity and ~100 nm ligaments [Elastic modulus is another important mechanical property and is required to calculate residual stress in freestanding beams, as seen in igaments . Elasticigaments . This meigaments . With thet al. extracted measured elastic modulus of ~9 GPa on dog-shape beams described earlier [As for measurements on freestanding np-Au beams, Lee  earlier , while v earlier ,54. Theset al. measured elastic modulus (~7–12 GPa) from the unloading segments of compression tests on nano-pillars with ~15 nm ligament sizes [et al. collected stress-free np-Au foils produced from alloy foils on elastomer blocks and captured characteristic buckling wavelength when the block is uni-axially compressed [The emergence of compression tests on np-Au micropillars contributed more elastic modulus values to the current literature. Volkert nt sizes . In addimpressed . They pret al. demonstrated that np-Au strips can withstand much higher reversible mechanical deformations without fracturing when wet than when dry [Most groups focused on yield stress and strength of np-Au, with the motivation of creating a low-density high-strength material. While np-Au films are notoriously brittle in macro scale, both SEM and TEM studies showed that ductile necking in severed ligaments demonstrate the ductile nature of np-Au ,54,58,71when dry .et al. performed nanoindentation studies on np-Au samples with ~42% porosity and ~100 nm ligaments and extracted yield stress of the porous network from hardness measurements to be ~145 MPa [i.e., relationship between physical and mechanical properties [et al. in compression studies of compact Au posts [et al. performed compression tests on micron-sized np-Au nano-pillars with ligament diameters of ~15 nm and ~36% porosity. Using foam scaling laws, they predicted that the 15 nm ligaments yield around ~1.5 GPa, which is close to the theoretical strength of gold [et al. [et al. conducted a comprehensive study on scaling equations of porous foams with various ligament sizes (10–900 nm) and porosities (20–42%), and reiterated that the yield strength is not only determined by porosity but also by ligament diameters [i.e., elastic modulus, yield stress) of FIB-prepared micropillars with small volume-to-peripheral surface area may be dominated by the surface damage [et al. reviewed the current literature stating that yield stress increases with decreasing ligament size and contrasted this notion with their studies on crack-free np-Au samples  demonstrating much lower yield stress values predicted by others [The majority of yield stress measurements utilized nanoindentation of bulk np-Au samples or compression tests on micropillars. Biener ~145 MPa . They nooperties ) under-poperties . A generAu posts , possiblAu posts . Volkert of gold —in agree [et al. . They coiameters . They moe damage . Others e damage . Jin et y others . They sui.e., residual stress, elastic modulus, yield strength) can be modulated via various techniques, which opens the door to development of advanced materials, such as low-density high-strength foams, as well as high-surface area low-elastic modulus coatings for chemo-mechanical sensors.The overarching significance elucidated by the mechanical characterization of np-Au foams has been to show that the mechanical properties  offer the additional flexibility in capturing surface topography such as roughness. Conventional measurement techniques are usually inadequate in determining thickness of freestanding microbeams. An  problem . Basical problem . WLI sys problem . Dynamic problem , can be et al. reviewed in situ nanoindentation of np-Au in TEM, where ligament deformation at length scales at ~10 nm in ~75–300 nm-thick np-Au films and underlying mechanisms were investigated [Energy dispersive spectroscopy, where the material is excited by electron beams and the characteristic emission in high energy bands is measured, has been a vital tool in characterizing the elemental composition of initial alloy and residual constituents in the final nanoporous film ,43. Dealstigated . The autTotal surface area, which made np-Au a very attractive material for sensor applications, can be estimated by gas adsorption methods  and elechttp://rsb.info.nih.gov/ij/index.html). Image processing of 2D morphologies has its own challenges as well. The first step of a typical image processing algorithm is the thresholding of a gray-scale image to produce a monochrome image. The central question here is picking a gray scale value  that accurately represents the separation between dark regions  and light regions . In determining this, the imaging parameters such as working distance, brightness, contrast, and electron acceleration energy should be consistent for each image. The selection of the threshold value is highly prone to subjective bias, which can be circumvented (at least kept consistent from image to image) by using automatic thresholding algorithms, such as ISODATA  [i.e., bias towards defining lighter pixels as pores) results in over-estimation of porosity and average pore size. Another challenge is the irregularity of pore shapes, which complicates the determination of a characteristic pore size. The “watershed” algorithm partitions the images into regions that are roughly circles [etc.). For quantitative analyses, approaches such as transmission electron tomography may be appropriate.Here, we will mostly focus on post-processing of SEM images for morphology analysis. Usually, the images acquired with SEM need to be post-processed to extract morphology parameters of interest. It is laborious to manually measure a statistically meaningful number of pores. It is more practical to use image analysis software for this purpose, such as ImageJ  . The conchnique) . Figure  circles , which cAlthough now a general understanding of porosity evolution exists, it is necessary to study the effects of stress generation during dealloying in structures under different mechanical constraints and the effects of multiple alloy constituents in pore morphology.Identifying the relationship between alloys constituents and resulting morphologies, particularly for alloy systems with multiple constituents, can assist in expanding the repertoire of nanoporous gold with distinct pore structures and enable a multitude of applications.The coarsening effect of thermal treatment on porosity evolution is well known, but the dynamics of the process are probed mostly theoretically. An interesting experiment may involve real-time observation of porosity evolution under thermo-mechanical stress using a scanning electron microscope with a heated stage to provide insight into routes of coarsening.Film delamination and cracking limit the successful integration of np-Au as functional coating. Most delamination problems during dealloying are a result of the absence of the adhesive layers between the np-Au layer and the substrate, compounded by residual stress accumulation due to volume contraction. Characterization of the interface strength and optimization of fabrication variables may mitigate such issues, thereby enabling synthesis of intact np-Au structures.Volume contraction during dealloying is widely observed and there are several methods to blunt its scale; however, it should be more systematically studied with particular attention to the effects of key parameters, such as electrolyte type, temperature, sample dimensions, and dealloying potential.Flow-through np-Au catalysts and molecular sieves are promising applications; however, the pressure-flow relationships as well membrane strength with respect to membrane architecture need to be established in order to implement this structure in such applications.We have presented a review of recent research in np-Au with an emphasis on fabrication and characterization techniques. Despite the rapidly growing knowledge in mechanical properties of np-Au, there are still unknowns with regard to fracture mechanics, the effects of alternate gold alloys on resultant pore morphology and mechanical properties, effects of sample geometry on pore evolution, and mass transport within the porous network. Here, we summarize some areas, in our opinion, that require further studies:The aforementioned questions will likely require the development of novel fabrication and characterization methods, as well as modeling approaches. We envision that combined efforts focusing on these issues will increase our understanding of np-Au and facilitate its implementation in a variety of applications. One such important application is the incorporation of np-Au for medical purposes, including immuno-sensors and neural electrodes. Such devices can be very powerful as point-of-care medical devices provided that their dependence our bulky external circuitry is prevented. In that aspect, we expect that microfluidic platforms would greatly benefit miniaturization of np-Au based sensors. The research at the interface of biology and np-Au is nascent. We foresee great potential in np-Au as a functional medical coating, which can be realized by research focusing on the biocompatibility of np-Au via tissue-material interaction studies. In addition, np-Au is a strong candidate for controlled drug delivery, due to its highly controllable pore morphology , which s


Four hypotheses were derived from Wojdylo’s work craving theory and Kuhl’s self-regulation theory: (H1) Work craving is associated with a combination of low self-regulation and high self-control. (H2) Work craving is associated with symptoms of psychological distress. (H3) Low self-regulation is associated with psychological distress symptoms. (H4) Work craving mediates the relationships between self-regulation deficits and psychological distress symptoms at high levels of self-control. Additionally, we aimed at supporting the discriminant validity of work craving with respect to work engagement by showing their different volitional underpinnings. Results of the two studies confirmed our hypotheses: whereas work craving was predicted by high self-control and low self-regulation and associated with higher psychological distress, work engagement was predicted by high self-regulation and high self-control and associated with lower symptoms of psychological distress. Furthermore, work styles mediated the relationship between volitional skills and symptoms of psychological distress. Based on these new insights, several suggestions for prevention and therapeutic interventions for work-addicted individuals are proposed.Work craving theory addresses how work-addicted individuals direct great emotion-regulatory efforts to weave their addictive web of working. They crave work for two main emotional incentives: to overcompensate low self-worth and to escape  negative affect, which is strategically achieved through neurotic perfectionism and compulsive working. Work-addicted individuals’ strong persistence and self-discipline with respect to work-related activities suggest strong skills in volitional action control. However, their inability to disconnect from work implies low volitional skills. How can work-addicted individuals have poor and strong volitional skills at the same time? To answer this paradox, we elaborated on the relevance of two different volitional modes in work craving: self-regulation  Work-addicted individuals–so called workaholics–experience that they are only really “alive”, when working . T). T2]). Researchers have traditionally portrayed workaholism as a phenomenon that manifests almost entirely in obsessive-compulsive symptoms: obsessive thinking about work  and a compulsive pattern of working behavior both behavioral and mental  argued addictive elements for better understanding the nature of work addiction. According to work craving theory (, t, t11], t theory , positive reinforcement as well as negative reinforcement. Specifically, the theory assumes that work-addicted persons become pathologically focused on pursuing reward from engaging in the behavior of compulsive working and neurotic standards: achieving positive outcomes such as self-worth, and social acceptance (positive reinforcement) and avoiding aversive outcomes such as negative emotions (negative reinforcement). Work-addicted individuals struggle with refraining from the behavior, experience intense craving, and have difficulty resisting; they have limited awareness of the problems that have emerged as a result of their behavioral addiction. Thus, according to the work craving theory, the phenomenon of workaholism is only dysfunctional. There is no functional form of workaholism .It should be noted that for convenience, we use the terms “work-cravers” and “work-addicted individuals” interchangeably for those individuals scoring relatively high on the Work Craving Scale (WCS), taking into account that the WCS has been analyzed as a continuous and not as a categorical variable . In conWojdylo et al.  providedintrojected regulation), engaged employees are working under the influence of intrinsic motivation, a positive connection with work, and the ability to deal with the demands of their jobs  . R. R15]. Rulation) .According to work craving theory, the emotion regulatory strategies of work cravers  imply that work craving should derive from low rather than high self-regulatory competencies. Indeed, Wojdylo, et al.  found thThese findings imply that work craving and work engagement may be associated with low versus high self-regulation competencies, respectively. This, in turn, inspires the question: How do work cravers manage to initiate and maintain their excessive work activities if they have poor volitional competencies? Their ability to focus on work-related goals suggests that work cravers also have strong volitional competencies.Self-regulation is conceptualized as a “democratic” mode of volition in which individuals form goals in accordance with their needs and preferences (self-determination), regulate emotions in a context-adequate manner (self-motivation and self-relaxation), and flexibly resolve action-related conflicts. It is self-supportive and fosters an overview and integration of own needs, wishes, and goals. Self-control, in contrast, is conceptualized as an “authoritarian” mode of volition in which individuals maintain a specific goal by suppressing any tempting alternatives, even if these alternatives are self-congruent. It is self-suppressive, focused on goal maintenance and self-discipline, and supported by high planning abilities for specific action steps.Kuhl’s theory of self-regulation provides a useful framework for understanding the volitional competencies of work cravers , 19], , , 21]. A. A21]. AWhereas self-regulation operates by activating the reward system, identification with goals, and self-motivation through positive emotions , self-control operates by activating the punishment system, conscious and depletive efforts, and self-motivation via negative emotions  . ). 19]). Healthy psychological functioning requires both modes because self-regulation and self-control are adaptive under different conditions and have different (dis)advantages , 20]. E. E20]. Estrong self-control and strong activation of the associated punishment system . Biebrich and Kuhl ). and Kuhl  found thH1) Work craving is associated with low self-regulation and high self-control. (H2) Work craving is associated with symptoms of psychological distress. (H3) Low self-regulation is associated with symptoms of psychological distress. Because of the mixed effects of self-control—positive short-term effects as well as negative long-term effects of (too much) self-control , . C. Cego fiIn light of these findings, one may wonder why work cravers keep on going. Previous findings show that obsessive-compulsive workers are avoidance motivated and try to prevent failure, guilt, shame, and anxiety , 38], , . At the It is noteworthy that our findings were obtained in two heterogeneous general population samples: workers with different occupations and from different work environments. Work craving theory does not specify work environments or job characteristics. Instead, work craving is assumed to develop as a function of personality and may be utilized as a coping mechanism in nonwork contexts, for example, in college or after retirement. Items may have to be adjusted to allow a valid assessment of work craving in such contexts. Thus, work craving theory takes a personality perspective on work addiction.In support of the discriminant validity of work craving, we found work engagement to have a completely different profile of volitional competencies: high self-regulation and high self-control. Furthermore, higher work engagement was associated with lower psychological distress and mediated the relationship between self-regulation competencies and health. Thus, work engagement is clearly a healthy work style. Engaged workers’ self-regulatory ability to integrate the multitude of personal needs, wishes, and goals may also contribute to successful work-life balance . Taken tNote that self-regulation and self-control show a significant positive correlation in our studies. This is because both are volitional competencies. Functionally distinct dimensions can correlate to the extent that they cooperate under certain conditions. In healthy individuals, self-control and self-regulation should cooperate whenever this seems reasonable, for example, when it makes sense to invest impulse control (as an example of self-control) after having made a self-congruent decision (as an example of self-regulation). Work engagement seems to indicate this type of healthy cooperation. Nevertheless, self-regulation and self-control are distinct volitional competencies because they may dissociate: one competency may be available in excess while the other is deficient. Work craving seems to indicate this type of dissociation and one-sided volitional action control (excessive self-control and deficient self-regulation).Our study has several limitations that can be addressed in future research. First, our findings are cross-sectional and do not allow us to draw causal conclusions. However, self-regulation and self-control are relatively stable dispositions that develop from early childhood onwards , 19]. T. T19]. TSecond, our outcomes are merely self-reports of psychological distress symptoms. In future studies, it would be informative to broaden the network of possible outcomes  and to extend measures to less obtrusive data . Third, we have concentrated on a personality perspective in our studies. It would be informative to assess work environment factors in order to explore their role in strengthening or ameliorating volitional competencies and/or work styles. For instance, employees’ self-control and work craving may be fostered in a company where the policy and its managers focus on the task and time pressure. In contrast, a company that values benevolence may employ more autonomy-supportive managers who foster employees’ self-regulation and work engagement. Future studies can verify if a more supportive work environment may compensate employee’s self-regulation deficits and function as a moderator at different positions in the model depicted in Finally, our findings have important practical implications. Forstmeier and Rüddel  have indS1 File(XLS)Click here for additional data file.S2 File(XLS)Click here for additional data file.


Leptomeningeal carcinomatosis (LC) is found in around 4% of patients with non-small cell lung cancer (NSCLC). The most common radiological finding of LC is diffuse leptomeningeal enhancement on contrast-enhanced brain magnetic resonance imaging (MRI). Herein, we report a novel brain MRI finding—non-enhanced, band-like, symmetric restricted diffusion along the anterior surface of the brainstem—of LC in four patients with NSCLC. We also identified three additional cases with similar MRI findings in a literature review. We hypothesized that the restricted diffusion along the anterior brainstem was caused by malignant cells concentrating in the cistern around the brainstem and infiltrating into the circumferential perforating arteries along the anterior brainstem surface, which then resulted in microinfarctions. Leptomeningeal carcinomatosis (LC) is found in around 4% of patients with non-small cell lung cancer (NSCLC), and it is associated with a median survival of only 5 months . Althoug3), elevated protein (376.9 mg/dl), elevated carcinoembryonic antigen (805.9 ng/dl), and a negative oligoclonal band. CSF cytology showed malignant cells. Serum anti-Ma2 antibodies and anti-glutamic acid decarboxylase antibodies were positive.A 39-year-old male smoker with diabetes mellitus was diagnosed with lung adenocarcinoma with epidermal growth factor receptor (EGFR) gene mutations in June 2011. He was treated with tumor resection and adjuvant chemotherapy (vinorelbine and cisplatin). Lung-to-lung metastasis developed in March 2012, after which he was treated with gefitinib. In July 2012, he presented with progressive dizziness, tinnitus, hearing impairment, seizures, and unsteady gait. A neurological examination revealed nystagmus, binocular diplopia, and ataxic gait. Brain MRI showed a non-enhanced band-like lesion along the anterior surface of the middle cerebellar peduncle, pons, and medulla with a high signal on fluid attenuation inversion recovery (FLAIR) imaging, and a restricted diffusion pattern on diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) mapping , and gefitinib. Four years later, the case presented with progressive dizziness, unsteady gait, and seizures. Brain MRI showed parenchymal metastasis at the left cerebellum and a non-enhanced symmetric band-like anterior brainstem lesion with a restricted diffusion pattern Figure . The casThe second case had progressively slurred speech, swallowing difficulties, and general weakness for 2 weeks before visiting our neurology clinic. Brain MRI showed non-enhanced symmetric band-like anterior brainstem lesions with restricted diffusion  or chemotherapy-related toxic encephalopathy may explain the brain lesions. Chemotherapy-related toxic encephalopathy was unlikely because different chemotherapy agents or TKIs were given in six cases, and one case was treatment-naive. PNS was also less likely because the brainstem findings did not fit any known PNS. Moreover, only one case had positive anti-Ma2 antibodies, and this case exhibited a poor response to intravenous steroid and intravenous immunoglobulin treatment. Considering that three cases had malignant cells in their CSF and another three cases had brain parenchymal metastasis, LC was the most likely etiology.via Virchow-Robin spaces. We speculate that the anterior brainstem restricted diffusion in our case series may have been caused by malignant cells concentrating in the CSF cistern around the brainstem and then infiltrating into the circumferential perforating arteries along the anterior brainstem surface, thereby resulting in microinfarctions.The most common brain MRI finding of LC is diffuse leptomeningeal enhancement . HoweverMost cases in our series had NSCLC with EGFR mutations, for which survival has been shown to be longer if treated with TKIs; however, the central nervous system was a frequent site of recurrence in these patients . The repIn conclusion, we reported a novel brain MRI finding with anterior brainstem restricted diffusion in patients with NSCLC. We believe that this is a new imaging finding of LC, and that it may be caused by high cellularity of the tumor cells on the brainstem or microinfarctions due to intracapillary tumor cell infiltration. Because brain MRI is an easily accessible and non-invasive diagnostic tool for LC, the identification of this unique imaging finding could be helpful in diagnosing LC. More case series are warranted and may provide further information about the risk factors and prognosis of patients with this imaging finding. Further studies with a larger number of cases and biopsy or autopsy findings may be able to elucidate the pathological features associated with this imaging finding and support our hypothesis.This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Written informed consent for case 1 has been obtained.C-YC: literature review, manuscript writing, figures making. C-YH: table making, literature review, manuscript writing. Y-HT and S-CC: imaging analysis. K-LL: paraneoplastic antibodies testing. C-EH and Y-HF: cases review. Y-CH: figures making, final manuscript review, and editing.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.


Natural killer (NK)/T-cell lymphoma is formally referred to as extranodal NK/T-cell lymphoma, nasal type (ENKTCL), in the 2008 and 2016 World Health Organization (WHO) classifications. NK/T-cell lymphoma, nasal type, is a rare but clinically important lymphoid neoplasm. It is the predominant type of extranodal lymphoma associated with the Epstein–Barr virus (EBV). NK/T-cell lymphoma is marked by a wide cytomorphological spectrum. The cytological findings may be so subtle that NK/T-cell lymphoma could possibly be easily overlooked. Here, we report a case of NK/T-cell lymphoma involving the sinonasal region with lymph node involvement. Fine needle aspiration of the neck lymph node and punch biopsy of the nasal mucosa were performed. The diagnosis of NK/T-cell lymphoma was confirmed based on pathological and immunohistochemical analyses, as well as in situ hybridization for EBV-encoded mRNA (EBER). The present case report underlines the importance of prompt clinicopathological assessment in suspected cases. The comparison of cytomorphologic features of NK/T-cell lymphoma in various specimens is presented. Extranodal NK/T-cell lymphoma, nasal type (ENKTCL), is a unique form of mature NK/T-cell lymphoma that is invariably associated with the Epstein–Barr virus (EBV) and exhibits a predilection for occurrence in certain ethnic groups. The disease is more prevalent among Asians and Native Americans as compared with the populations in the western countries –3. In thA 39-year-old man presented with nasal obstruction accompanied with foul-smelling discharge for the last 1 month. In addition, he complained of headache, poor appetite, and bilateral palpable neck masses. On physical examination, firm and nontender lymph nodes were found over bilateral level II regions. Fiberoptic endoscopy of the left nasal cavity showed mucosal erosions and whitish exudates coating the mucosal surface . LaboratUltrasound-guided FNA cytology (FNAC) was performed by standard technique using a 22-gauge needle for the left neck lymphadenopathy. One air-dried and one alcohol-fixed smear, as well as liquid-based cytology  specimen, were prepared for cytological evaluation. The cell sample was fixed in CytoRich fixative, and additional cell block was also prepared from the remnants of LBC specimen. The air-dried smear was stained with Liu's stain. The alcohol-fixed smear was stained with Papanicolaou stain.FNAC revealed dispersed hypercellular pattern, with a polymorphous mixed population of small-, intermediate-, and large-sized lymphoid cells. Follicular center cells, histiocytes (including tingible body macrophages), and lymphohistiocytic aggregates were not found. Plasma cells and eosinophils were scant in numbers. In Liu's stain slide, lymphogranular bodies were well recognized in the background. There were medium-to-large atypical cells, which exhibited irregular nuclei with one to several nucleoli and pale blue or clear cytoplasm. Cytoplasmic projections with tongue-like protrusions and fine or coarse cytoplasmic azurophilic granules were also present . MitoticThe biopsy specimen of the left nasal lesion showed diffuse mucosal necrosis with the presence of granulation tissue and dense cellular infiltrates. Atypical lymphoid cells of varying sizes with prominent nuclear irregularity and pale or clear cytoplasm were also observed. Large atypical lymphoid cells, significant apoptosis, and scattered mitotic figures were noticed. The neoplastic cells exhibited an angiocentric growth pattern, as well as angiodestruction with mural fibrinoid necrosis and hemorrhage . PseudoeNatural killer (NK)/T-cell lymphoma is formally referred to as extranodal NK/T-cell lymphoma, nasal type (ENKTCL), in the 2008 and 2016 World Health Organization (WHO) classifications. NK/T-cell lymphoma located in the nasal region is the prototype for this lymphoma, which was formerly referred to as polymorphic reticulosis. The disease typically affects the nose and facial midline and exhibits aggressive destruction caused by cytotoxic activity, due to which it was classified as a lethal midline granuloma . NK/T-ceε and negative staining for surface CD3. Lymphoma cells exhibit positive expression of cytotoxic granule proteins such as granzyme B, TIA-1, and perforin. Other markers such as CD43 and CD45RO may be positive; positive expression of CD30 may also be found in some cases [The immunophenotype of typical NK/T-cell lymphoma is marked by positive staining for CD2, CD56, and cytoplasmic CD3To the best of our knowledge, the cytological features of NK/T-cell lymphoma, nasal type, are not well established in the published literature –14. NK/TCytological diagnosis based on FNAC specimens is typically straightforward in case of certain subtypes of lymphoma, such as the conventional diffuse large B-cell lymphoma and Burkitt's lymphoma, which are marked by a monotonous tumor cell population . In contCytological differential diagnosis of NK/T-cell lymphoma from other types of lymphoma is a challenge. Diffuse large B-cell lymphoma typically exhibits large cells with rounded noncleaved nuclei. Grade 1 or grade 2 follicular lymphoma shows predominantly medium-sized centrocytes with irregularly cleaved nuclei, centroblasts with noncleaved nuclei, and follicular aggregates on Pap staining. In contrast to the Reed–Sternberg cells in Hodgkin's lymphoma, the atypical lymphoid cells of NK/T-cell lymphoma do not contain large, inclusion-like, eosinophilic nucleoli , 13. NK/In the current case, FNAC of the lymph node shows a heterogeneous population of small-, medium-, and large-sized lymphoid cells, with the lack of reactive cellular components such as follicular center cells or lymphohistiocytic aggregates. The medium- to large-sized lymphoid cells exhibit readily discernible nuclear atypia with an irregular nuclear outline. The cellular shrinkage is obvious in LBC specimen, while the cytoplasmic protrusion is more easily visible than conventional smears. The degree of nuclear atypia far exceeds that of reactive changes. Lymphoma cells with elongated and twisted nuclei, recapitulating the cellular morphology seen in biopsies of the usual nasal-type NK/T-cell lymphoma, are common . The NK/In a recent case series of NK/T-cell lymphoma, nasal type, in Taiwan, a total of 73 cases were retrospectively investigated with a median age of 54 years and a male-to-female ratio of 2.0 : 1 . The uppAlthough ENKTCL is sensitive to radiotherapy, it shows a poorer response to chemotherapeutic agents than other lymphomas due to expression of p-glycoprotein . At presDefinitive cytomorphologic diagnosis of lymphoma is possible but can be challenging in routine cytology specimens. Review of a previous case series report showed that a diagnosis of NK/T-cell lymphoma is possible on FNA or body fluid cytology, given adequate ancillary testing support. Cell block material, if available, is an important adjunct to diagnosis.Immunocytochemical study, as well as more sophisticated techniques, can be readily applied to cell block paraffin sections. Consideration of clinical features, cytology, and/or histomorphology and determination of immunophenotype on immunohistochemical examination or flow cytometry are useful for arriving at a definitive diagnosis.


The processes underlying persistence and remission of attention-deficit/hyperactivity disorder (ADHD) are poorly understood. We examined whether cognitive and neurophysiological impairments on a performance-monitoring task distinguish between ADHD persisters and remitters.On average 6 years after initial assessment, 110 adolescents and young adults with childhood ADHD  and 169 age-matched control participants were compared on cognitive-performance measures and event-related potentials of conflict monitoring (N2) and error processing (error-related negativity and positivity) from an arrow flanker task with low-conflict and high-conflict conditions. ADHD outcome was examined with parent-reported symptoms and functional impairment measures using a categorical (DSM-IV) and a dimensional approach.p < .05). ADHD remitters differed from persisters and were indistinguishable from control participants on the number of congruent (low-conflict) errors, reaction time variability, error-related negativity, and error-related positivity . Remitters did not differ significantly from the other groups on incongruent (high-conflict) errors, mean reaction time, and N2. In dimensional analyses on all participants with childhood ADHD, ADHD symptoms and functional impairment at follow-up were significantly correlated with congruent errors, reaction time variability, and error-related positivity .ADHD persisters were impaired compared with controls on all cognitive-performance and event-related potential measures (all Cognitive and neurophysiological measures of attention-vigilance and error detection distinguished ADHD remitters from persisters. These results extend our previous findings with other tasks and indicate that such measures are markers of remission and candidates for the development of nonpharmacological interventions. The identification of cognitive and neural processes underlying the trajectories of persistence and recovery from childhood-onset disorders during the transition to adulthood has the potential to prevent negative long-term outcomes , 2. AtteIt has been proposed that the cognitive processes associated with persistence of ADHD across development may be separate from those linked to the remission of the disorder . HoweverThe assessment of neurocognitive processes using cognitive and brain activity data may allow a deeper understanding of the developmental trajectories of ADHD. Our recent investigation of adolescents and young adults with childhood ADHD assessed on a range of cognitive, event-related potential (ERP), and electroencephalography (EEG) measures found that ADHD remitters differed from persisters, but not from control participants, on preparation-vigilance measures  and actigraphic data on movement. Executive-function processes of inhibition and working memory , instead, were not sensitive to ADHD persistence or remission, as ADHD remitters showed an intermediate pattern between persisters and control participants, without significant differences from either group . FurtherNeurocognitive impairments in ADHD include deficits in performance monitoring, an essential cognitive ability in goal-directed behavior to monitor ongoing performance and to adjust response selection , 17, 18.In ADHD, N2 attenuation in the flanker task has been reported in children and adults with ADHD , 25, 26,In the present study, we aimed to extend our recent findings  by invesThe sample consists of 279 participants, who were followed up on average 5.8 years (SD = 1.1) after initial assessments: 110 had a diagnosis of DSM-IV combined-type ADHD in childhood (10 sibling pairs and 90 singletons) and 169 were control participants (76 sibling pairs and 17 singletons) , 31. ParF = 11.34, p < .01) and functional impairment  than those who were not taking medication. However, the proportion of participants on medication did not differ between ADHD persisters and remitters . A 48-hour ADHD medication-free period was required prior to assessments. Three ADHD persisters (3.4%) were also on antidepressant medication, but for ethical reasons they were not asked to stop taking them. These participants were included in all analyses as their exclusion did not alter the results. Parents of all participants gave informed consent following procedures approved by the London-Surrey Borders Research Ethics Committee (09/H0806/58).Among those with childhood ADHD, 87 (79%) continued to meet clinical (DSM-IV) levels of ADHD symptoms and impairment (ADHD “persisters”), whereas 23 (21%) were below the clinical cut-off (ADHD “remitters”) . Among AThe Diagnostic Interview for ADHD in adults (DIVA)  was condAn estimate of IQ was derived with the vocabulary and block design subtests of the Wechsler Abbreviated Scale of Intelligence .The task was an adaptation of the Eriksen Flanker paradigm designed to increase cognitive load as used in previous studies , 25, 36.The EEG was recorded from a 62-channel DC-coupled recording system (extended 10–20 montage), using a 500-Hz sampling rate, impedances under 10 kΩ, and the FCz electrode as the recording reference. The electro-oculograms were recorded from electrodes above and below the left eye and at the outer canthi. EEG data were analyzed using Brain Vision Analyzer 2.0 . Raw EEG recordings were down-sampled to 256 Hz, rereferenced to the average of all electrodes (turning FCz into an active channel), and filtered using Butterworth band-pass filters . All trials were visually inspected for electrical artifacts or obvious movement, and sections of data containing artifacts were removed manually. Ocular artifacts were identified using the InfoMax independent component analysis algorithm . SectionAnalyses of ERPs of performance monitoring were restricted to incongruent trials, as the task used in this study is known to elicit strong N2, ERN, and Pe components in high-conflict, but not in low-conflict, conditions , 25, 36.d effect sizes are presented along with means, SDs, and test statistics for the group analyses , condition , and group by condition interaction using random intercept models in Stata  to control for genetic relatedness of the sibling pairs in a repeated-measures design. A random intercept model was also run to test the effect of group, scalp site  and group by site interaction on the N2. ERN and Pe were analyzed with regression models with dummy variables to identify overall group effects, controlling for sibling relatedness with the “robust cluster” command in Stata. Age correlated significantly with several of the cognitive-ERP measures  and was analyses , where 0analyses . PearsonBecause ADHD persisters had lower IQs than remitters did  14), an, an14), An overall group effect emerged on all cognitive-performance and ERP measures . Post hon = 63) and remitters (n = 20) became a trend for the ERN and nonsignificant for the Pe. Given the small female sample sizes  and the discrepancy in the size of male and female groups, sex differences were not directly tested. However, the effect sizes in the male-only sample  were comparable or only slightly reduced compared with those of the full sample. Remitters significantly differed from control participants on incongruent MRT, congruent RTV, and incongruent RTV, but not on incongruent errors. All other results remained unchanged. For further details, see the Controlling for IQ, group effects on MRT in both conditions and N2 at FCz were nonsignificant . Differen = 110), both ADHD symptoms and impairment at follow-up significantly correlated with the Pe  and neurophysiological error processing (ERN and Pe) compared with persisters. Attention-vigilance measures and conscious error processing were also associated with the continuum of ADHD symptoms and impairment at follow-up. Conversely, measures of executive control (incongruent errors), speed of processing (MRT), and neurophysiological conflict monitoring (N2) did not distinguish remitters from persisters, and thus they were not sensitive to remission or persistence of the disorder. Processes of attention-vigilance and neurophysiological error processing can be markers of remission from ADHD and may be sensitive to the effects of training or compensatory mechanisms.RTV, measuring intraindividual variability in reaction time, and number of congruent errors in the low-conflict condition distinguished ADHD remitters from persisters, but not from control participants, and were also correlated with continuous ratings of ADHD symptoms and impairment. Impairments in such measures in the congruent condition of the flanker task may result from lapses in attention and index attention-vigilance processes. Neurophysiological measures of error processing (ERN and Pe) showed the same association with ADHD remission. Conscious error processing (Pe) also correlated with the continuous ADHD symptoms and functional impairments at follow-up. Of note, the group differences observed on this peak-to-peak ERN were likely explained by the voltage change from the PNe to the negative ERN peak see the . This mePrimary analyses did not control for IQ, as lower-mean IQ in ADHD samples represents one of multiple cognitive processes underlying ADHD pathophysiology , 44, andThe present study extends the findings in our previous investigation that used a cued continuous performance test (CPT-OX), a four-choice reaction time task, and Wechsler Abbreviated Scale of Intelligence measures of IQ and digit span . Attentid = 0.44–0.75) between persisters and remitters in measures representing markers of remission, but small or negligible effect sizes (d = 0.02–0.28) in measures not sensitive to ADHD outcome at follow-up, suggesting this study had sufficient power to detect the major correlates of remission with the current sample sizes. Furthermore, when we repeated the analyses for male participants only, differences between remitters and persisters in the ERN and Pe were reduced. However, the small sample of female participants did not allow a direct examination of sex differences. Future studies that include a higher number of female participants are needed to further investigate these processes also in females. Finally, our sample included young adults as well as adolescents, who are still undergoing rapid cortical maturation. Although we controlled for age in all analyses, future follow-up assessments with participants having reached adulthood and when more ADHD participants may have remitted could clarify matters further.A limitation of this study is that, despite the large sample size, the low ADHD remission rate at follow-up resulted in a relatively small group of remitters. Therefore, we could not rule out the possibility that some nonsignificant differences between remitters and other groups could be due to low power. However, we observed medium-to-large effect sizes (Overall, we report that attention-vigilance and neurophysiological error processes were impaired in ADHD persisters but not in remitters and may be sensitive to compensatory mechanisms in those who remit from the disorder. These processes may be targets for nonpharmacological interventions or behavioral training aimed at alleviating some of the long-term outcomes of ADHD. Conversely, cognitive measures of executive control, speed of processing, and conflict monitoring were not sensitive to ADHD remission/persistence. Considering the importance of using a broad range of cognitive and neural measures in investigating the mechanisms underlying neurodevelopmental disorders , our cog


Drosophila spermathecae consist of class-III secretory units, in which each secretory cell (SC) discharges its products to the central lumen through an end-apparatus and a canal. Secretory unit formation in Drosophila spermathecae utilizes a fixed cell lineage, in which each secretory unit precursor (SUP) divides to produce one pIIb cell and one pIIa cell. The former differentiates into an apical cell (AC), whereas the latter divides again to produce an SC and a basal cell (BC). It is unclear how each cell acquires its identity and contributes to secretory unit formation. Here, we demonstrate that Notch signaling is required and sufficient for the specification of lumen epithelial precursors , pIIb (vs. pIIa), and SCs (vs. BCs) sequentially. To our surprise, Notch activation in LEPs and SCs apparently utilizes different ligand mechanisms. In addition, Notch signaling both suppresses and activates transcription factors Hindsight (Hnt) and Cut during spermathecal lineage specification, supporting the notion that Notch signaling can have opposite biological outcomes in different cellular environments. Furthermore, LEP-derived epithelial cells (ECs) and ACs show distinct cellular morphology and are essential for securing secretory units to the epithelial lumen. Our work demonstrates, for the first time, the dynamic role of Notch signaling in binary cell fate determination in Drosophila spermathecae and the role of ECs and ACs in secretory unit formation.Spermathecae are glandular organs in the insect female reproductive tract that play essential roles in insect reproduction; however, the molecular mechanism involved in their development is largely unknown.  Drosophila have shown that glandular secretions from spermathecae and parovaria act to attract, nourish, and protect sperm by creating an appropriate environment  seems not to be involved in Notch signaling in the spermathecal lineage. Interestingly, Notch ligand Delta (Dl) is required for LEP specification, but not for SC fate. By manipulating Notch signaling to transform cell fate, we demonstrate that ECs and ACs are essential to secure the secretory unit to the epithelial lumen and that each cell in the spermathecal lineage shows distinct cellular morphology consistent with their biological functions.Notch signaling is an evolutionarily conserved signaling pathway, which can be activated ligand-dependently or ligand-independently . Notch sSu(H)GBE-lacZ (Su(H)GBE-Gal4 , UAS-Su(H)DN , UAS-NICD (BDSC), RNAiUAS-Ser , and UAS-eGFP (BDSC).Flies were reared on standard cornmeal–molasses food at 25°, unless otherwise indicated. GBE-lacZ  and Su, and only one against homeobox transcription factor Cut showed a unique expression pattern in the spermathecal head at 48 hr APF  is required for the LEP fate. We used lz-Gal4 to drive RNAiUAS-N expression in all gland precursors, and found that N knockdown did not disrupt the formation of the double layer in the middle region of spermathecal heads; however, expression of Lz was absent in the apical layer of N-knockdown vs. control spermathecae . As this result indicated a loss of LEP identity, we next tested whether the apical layer in N-knockdown spermathecae gained the SUP fate (indicated by Hnt and Cut expression). In contrast to basal expression of Hnt and Cut in control spermathecae, Hnt and Cut were expressed in all cells of the middle region of N-knockdown spermathecae, indicating that the apical layer did indeed gain the SUP fate . Therefore, loss of N led to switch LEP fate into SUP fate.Since Notch signaling is activated in LEPs at the apical side of the spermathecal head , we testDNSu(H)] in gland precursors. Overexpression of DNSu(H) blocked Lz expression but induced Hnt and Cut expression in the apical layer at 26 hr APF ] by overexpressing a dominant-negative form [6 hr APF , suggestNotch intracellular domain (NICD) in gland precursors using lz-Gal4. NICD overexpression in gland precursors prevented spermatheca morphogenesis, arresting spermathecae at around 14 hr APF . To overcome this problem, we utilized 51B02-Gal4, which is expressed in the middle region of the spermathecal head prior to expression of Hnt and Cut and continues to be expressed at 48 hr APF , middle pIIb cells (Cut+), and basal pIIa cells  or Cut− cells (presumably pIIa cells), both of which are Hnt positive  and a BC (Hnt−), indicating that a flip-out event occurred in a pIIa cell  were composed of two BCs rather than an SC and a BC . In total, 17.4% of N-knockdown clones had an SC-to-BC transformation (DNSu(H) overexpression showed an SC-to-BC transformation  of the two-cell clones were two-BC clones and 26 out of 26 (100%) of the three-cell clones consisted of an AC and two BCs (DNSu(H)-overexpressing clones had an SC-to-BC transformation . At this time point, 25% of control SC clones consisted of two cells (an SC and a BC) and 75% consisted of three cells . Altogether, our data suggest that Notch signaling is required and sufficient for SC fate during the SC–BC binary cell fate determination.To determine whether Notch signaling is sufficient for SC fate, we induced Cs Hnt+;  or two SCs Hnt+; , indicatectively . To deteNICD-overexpressing clones induced at 24 hr APF occasionally consist of two cells with faint Hnt and GFP, presumably ACs . The remaining clones were composed of four or five cells, an AC, an SC, a BC, and one or two ECs, indicating a flip-out event in a gland precursor  or two ACs and one EC . The latter clones were derived from gland precursors and had a pIIa-to-pIIb transformation. In total, 56% of NICD-overexpressing clones had a pIIa-to-pIIb transformation when induced at 20 hr APF . This is similar to the clones induced at 24 hr APF  and 11% four- or five-cell clones with one or two ECs . Occasionally, two of the clone cells were slightly smaller than the other two (File S2); this was likely a gland precursor clone that produced two SUPs and subsequently eight BCs. In total, we observed that 59% of N-knockdown clones had a pIIb-to-pIIa transformation (DNSu(H) was overexpressed, although less frequently . These data suggest that Notch signaling is required for pIIb fate specification.To test whether induction of clones at an earlier time point could increase the pIIb-to-pIIa transformation rate, we induced clones at 14 hr APF. This experimental scheme resulted in 76% three-cell clones (SUP clones) and 24% four- or five-cell clones (P0 clones) in the control group . In contwn group , all of ther two ; this liormation . We obseN-knockdown clones were rarer when induced at 14 hr APF (22 clones from 47 RNAiN spermathecae vs. 39 clones from 14 control spermathecae). We frequently observed four-cell N-knockdown clones detaching away from the central lumen  as N-knockdown spermathecae at 26 hr APF , pIIb (vs. pIIa), and SCs (vs. BCs). This likely occurs in parovaria, as we frequently observed a similar cell fate transformation in parovaria when we manipulated Notch signaling. In contrast, it is surprising to us that Notch activation in LEPs and SCs apparently utilizes different ligand mechanisms, as knocking down Dl using the same RNAi construct is able to block LEP specification but not SC specification. Since Ser knockdown is also not required for SC specification, this may indicate that SCs utilize a ligand-independent mechanism to activate Notch signaling (Through lineage tracing, we have previously determined the solution ; howeversolution . Inspire lineage , we inveignaling . AlternaIt is still unknown how the asymmetric cell fate and Notch signaling are established in the spermathecal lineage and whether the same machinery for asymmetry is involved in the spermathecal lineage as in the sensory lineage, such as Numb and Neuralized . The facThe Notch pathway is evolutionarily conserved, plays pleiotropic roles in multiple organ systems, and frequently results in completely opposite biological consequences in different organs or different cell lineages within the same tissue . It is qIn comparison to class-I and class-II SCs, class-III SCs have the most complicated structure and discharge their secretion through a cuticular end-apparatus and canal . Most ofDrosophila spermathecae are built through a three-cell unit, including an AC, a BC, and an SC. No cilium is involved in the formation of this secretory unit (In contrast to cockroach tergal glands, secretory units of ory unit , but theory unit  and the ory unit . It remawww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.040212/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


While effective connectivity  between brain areas has been investigated in chronic users of cocaine as they view cocaine pictures cues, no study has examined EC while they take part in a resting-state scan. This resting-state fMRI study aims to investigate the causal interaction among brain areas in the mesocorticolimbic system (MCLS), which is involved in reward and motivation, in cocaine users (vs. controls).Twenty cocaine users and 17 healthy controls finished a structural and a resting-state scan. Mean voxel-based time series data were obtained from brain regions of interest (ROIs) from the MCLS, and were input into a Bayesian search algorithm called IMaGES.The causal interaction pattern was different between the two groups. The feed-forward pattern found in cocaine smokers, between 7 ROIs of the MCLS during resting-state , was absent in controls. That is, the subcortical VenStri area had a causal influence on four cortical brain areas only in cocaine users.During the resting-state scan, the VTA of cocaine smokers abstinent for at least 72 hours, but not controls, begins causal connections to limbic, midbrain, and frontal regions in the MCLS in a feed-forward manner. Following replication, further studies may assess if changes over time in EC during resting-state predict cocaine treatment efficacy and outcome. The mesocorticolimbic system (MCLS) has been linked to reward, motivation and goal-directed behavior. It contains the brain structures, namely, the ventral striatum (nucleus accumbens), extended amygdala, hippocampus, anterior cingulate, prefrontal cortex, and insula ,2. DrugsUsing functional magnetic resonance imaging (fMRI) technique, resting-state functional connectivity  has been examined on MCLS in cocaine users . Also, aIn this research, we investigated the causal interactions among all MCLS brain areas while participants’ brains were scanned at rest. This approach is valuable in terms of understanding how communication among MCLS brain areas changes due to neuroadaptation pertinent to long-term cocaine exposure. In the context of the cue-reactivity study, we collected resting-state fMRI data from non-treatment seeking cocaine users who were abstaining from cocaine for three days and similarly-aged controls. This research used IMaGES ), midbrain ), and frontal areas , medial frontal cortex (MFC), anterior cingulate cortex (ACC), dorsolateral prefrontal cortex (DLPFC)) in a feed-forward manner  during rIn a research done by Ray et al.,  each parImaging data were collected using a 3T Siemens Trio head-only fMRI scanner equipped with a standard Siemens head coil. While participants visually fixated on the cross, T2*-weighted echo planar images were acquired  covering the entire brain. A sagittal T1-weighed structural scan  was acquired in order to co-register it with the fMRI data .The nine bilateral ROIs included: VTA, HIPP, VenStri, OFC, MFC, ACC, DLPFC, amygdala, and insula .For imaging data pre-processing procedure. Time series data for each ROI for each participant in each group were fed to the IMaGES program. IMaGES produced two outputs: one for the cocaine smoking group and one for the controls. Unlike other EC measures, such as Granger Causality or DCM, IMaGES models directionality of the edges. IMaGES are based on regression, not correlation. A detailed explanation of the method and its approach to causality can be found in Ramsey et al (2010).Unpaired t-test results showed that the mean frame-wise displacement was not significantly different between the cocaine and control groups (p =0.8120).IMaGES showed that for smokers at resting-state , the VTAIn controls , HIPP, bThus, the causal interaction pattern found between the two groups was different, providing evidence for different underlying processing models during resting-state ,16. The Overall, the purpose of this analysis was to use IMaGES to compare EC among all MCLS brain areas in chronic users of cocaine to that of healthy controls during resting-state. In accordance with our hypothesis, for cocaine smokers the VTA was connected to limbic (HIPP), midbrain (VenStri), and frontal areas in a feed-forward manner during resting-state . Differences in the IMaGES graphs in the cocaine group compared to the control group required different solutions, which supported the underlying model of proposed differences during resting-state ,16.The present study extends previous research by Ray and colleagues 2016) by establishing that cocaine smokers’ VTA initiates a feed-forward causal interaction between the 7 regions in the MCLS at resting-state. It is noteworthy that the present findings are consistent with Ray and colleagues (2016) in that the VTA showed a direct activation upon HIPP during resting-state. However, the spectral DCM model in Ray et al. (2016) could not reveal the feed-forward connections between VTA, limbic, midbrain, and frontal areas, as identified in the current IMaGES analysis. Also, the current results are similar to findings in the same cocaine individuals during the cocaine cue exposure task [6 by estaThe direct activation of VTA on HIPP and on insula in chronic cocaine users is consistent with Jasinska et al.  who propIn the present control participants, the HIPP initiated three feedforward causal pathways  and therefore unlikely to have affected the results. Second, since we only had five female smokers, possible sex differences in EC during rest could not be investigated. Third, since amygdala ROI time series signal was correlated with the time series of multiple ROIs, IMaGES could not produce output using all nine ROIs. Amygdala had to be excluded from the IMaGES analyses. Even with these constraints, this research serves as a model of EC among MCLS ROIs at rest in chronic cocaine smokers.This research is the first demonstration in chronic cocaine smokers (but not in controls) that VTA initiates causal connections to limbic, midbrain, and frontal brain regions in MCLS in a feed-forward manner during resting-state scan. If these results are replicated, future research may assess if changes over time in EC during resting-state predict cocaine treatment efficacy and outcome.


FtsZ gene—which encodes a tubulin homolog responsible for the formation of the chloroplast inner division ring (Z ring)—in the Arabidopsis genome in 1995, many components of the chloroplast division machinery were successively identified. The knowledge of these components continues to expand; however, the mode of action of the chloroplast dividing system remains unknown , owing to the complexities faced in in planta analyses. To date, yeast and bacterial heterologous expression systems have been developed for the reconstitution of Z ring-like structures formed by chloroplast FtsZ. In this review, we especially focus on recent progress of our bacterial system using the model bacterium Escherichia coli to dissect and understand the chloroplast division machinery—an evolutionary hybrid structure composed of both bacterial (inner) and host-derived (outer) components.Plant chloroplasts originate from the symbiotic relationship between ancient free-living cyanobacteria and ancestral eukaryotic cells. Since the discovery of the bacterial derivative  Arabidopsis thaliana  . Th. ThArabili cells .E. coli reconstitution system has now become one of the many useful heterologous expression systems for studying the FtsZ-centered chloroplast division machinery. Although in planta analysis is the best way to comprehensively examine the components employed in chloroplast division, heterologous expression systems lacking such native division-related contributors also provide important insights into inherent properties of the introduced component(s). In general, microbial manipulations have practical advantages, such as rapid growth, axenic culture, and genetic accessibility. Yeast systems allow the advantage of a eukaryotic environment completely lacking plant derivatives. The advantages of bacterial systems are as follows: (i) bacteria is an evolutionary progenitor of chloroplast and bacterial cytosol, wherein the Z ring-centered division system still works and is topologically equivalent to chloroplast stroma; and (ii) bacteria—especially E. coli—are distantly related to cyanobacteria, and partly lack the homologs of chloroplast division-related proteins. Indeed, using the E. coli system, we reconfirmed the negative regulation of the chloroplast FtsZ2 filaments by plant-specific ARC3 and found that this regulation was also effective for the membrane-tethered one (Z ring-like structures) [E. coli [E. coli, which creates minicells but also shows normal- and long-sized cells, as in the case of AtFtsZ2 expressed in the ftsZ-depleted E. coli strain [E. coli systems into in planta results is important, and will continue to accelerate the research on chloroplast division. The uctures) . We alsouctures) . The comuctures) ,5. Besid[E. coli ,33,34,82i strain ,83. By ci strain ,77,84,85


Additionally, we studied cortical thinning and global and local measures of hippocampal atrophy as known key imaging markers for Alzheimer's disease. Apart from bilateral striatal volume reductions, no morphometric alterations were found in cognitively stable patients. In contrast, we identified shape alterations in striatal and thalamic regions in future converters at baseline and at time of conversion. These shape alterations were paralleled by Alzheimer's disease like patterns of left hemispheric morphometric changes  in future converters at baseline with progression to similar right hemispheric alterations at time of conversion. Additionally, receiver operating characteristic curve analysis indicated that subcortical shape alterations may outperform hippocampal volume in identifying future converters at baseline. These results further confirm the key role of early cortical thinning and hippocampal atrophy in the early detection of Alzheimer's disease. But first and foremost, and by distinguishing future converters but not patients with stable cognitive abilities from cognitively normal subjects, our results support the value of early subcortical shape alterations and reduced hippocampal subfield volumes as potential markers for the early detection of Alzheimer's disease.Efficacy of future treatments depends on biomarkers identifying patients with mild cognitive impairment at highest risk for transitioning to Alzheimer's disease. Here, we applied recently developed analysis techniques to investigate cross-sectional differences in subcortical shape and volume alterations in patients with stable mild cognitive impairment (MCI) ( Recent findings have demonstrated that beta-amyloid can be effectively removed from the brain, which may have a beneficial effect on cognitive function  allows the quantification of brain atrophy and represents a key imaging marker for the early detection of AD. Specifically, reduced gray matter volume in medial temporal lobe regions including the hippocampal formation may precede the clinical onset of AD by 10 years  as well as in MCI with future conversion to AD at their baseline and conversion timepoints. In this manner we were able to elucidate the potential value of subcortical shape measures and their ability to improve the identification of future converters to AD. Further, we use cortical and hippocampal measures to demonstrate anatomical trajectories of the subjects under study, thereby confirming their role in the early detection of AD.We expected stable MCI subjects to show no morphometric alterations typically related to AD, such as hippocampal atrophy or cortical thinning when compared with HC. In contrast, we expected morphometric alterations in accordance with known AD related histopathological processes in future converters at baseline and—more pronounced—at time of conversion. Specifically, and based on the pattern of AD related neurofibrillary tangle formation , and subjects with future cognitive worsening and conversion to probable AD  within a 2-year time frame during follow-up. Additionally, data from the MCI-CB group at time of conversion was obtained . Inclusion criteria for HC were: stable cognitive health ascertained by clinical work up and neuropsychological testing during an approximately 2-year follow-up. Exclusion criteria were: MRI exclusion criteria, left-handedness, evidence for abuse of alcohol and drugs, psychiatric, neurological or significant other system diseases. Three groups of HC were identified for group wise age and gender matching with MCI-S, MCI-CB, and MCI-CC, and MRI data was acquired following the description in section Magnetic Resonance Image Acquisition. The final demographic details are presented in Table For the present study, inclusion criteria for MCI subjects were: amnestic MCI (single or multiple domain) diagnosis and availability of MRI data at baseline and follow-up. Exclusion criteria were: left-handedness, significant medication or drug abuse as well as clinically significant neurological and psychiatric or internal disease that may affect cognition, MRI findings of infarction or other focal lesions, multiple lacunes or lacunes in critical memory structures. A total of 33 baselines from subjects with amnestic MCI were considered for the present study. The population was stratified into subjects with stable cognitive abilities during an approximately 2-year follow-up , slice thickness: 1 mm, total scan time: 15 min.All (MRI) were performed on the same 1.5 Tesla Phillips Achieva scanner using an 8-element head coil. Whole-brain high-resolution 3D TSegmentation of the striatum, thalamus and thalamic nuclei was performed using a recently developed label-fusion-based segmentation method that had previously proven its high accuracy . Briefly, the native images were linearly registered to the symmetric ICBM 152 template  indicate premorbid brain volume, and thus are often used to adjust volumes for subsequent volume analyses. When examining volume reductions in neurodegenerative disease, considering ICV allows for estimation of atrophy caused by neurodegenerative mechanisms rather than by interindividual differences in head size and brain morphology.In the present work, the FreeSurfer pipeline (version 5.1.0) was used to calculate total intracranial volume (eTIV) representing an estimate for ICV as described in Buckner et al. . BrieflyU-Test. Pearson's chi-square test was used for categorical variables. Tests were performed with a significance level of p < 0.05. Between-group differences in volumetric raw data  were examined by including age and gender as covariates in the multiple linear regression models. These analyses were repeated by using volumes relative to eTIV (volume/eTIV*100) in order to adjust volumes for differences in head size. P-values resulting from volume analyses were adjusted for multiple testing by using Bonferroni-Holm correction . The same models were performed for investigating between-group shape and cortical thickness differences. Vertex-wise analyses results are reported on a q-value corrected for multiple testing, using a false discovery rate (FDR) of 10% as in previous publications in our group , the average of the displacement values in mm was computed for each subject. This was done separately for each structure and hemisphere. Statistical analyses were performed with IBM SPSS statistics 21 and RMINC package , an image analysis software library developed for the R statistical environment (http://www.r-project.org).Group comparisons of demographic and conitive data were applied using analysis of variance (ANOVA) or Mann-Whitney Descriptive statistics for demographic information and cognitive data is listed in Table p-values were similar when using raw volumes instead of volumes relative to eTIV  after correction for multiple testing. With regard to thalamic subnuclei, and although statistically significant, effects of volume reductions in bilateral VP in MCI-CB  did not survive the correction for multiple testing. Significance and p-values were similar when using raw volumes instead of volumes relative to eTIV. However, some of the right hemispheric differences in hippocampal subfield volumes did not quite achieve the level of significance (CA1 p = 0.057). Additionally, and although statistically significant, two right hemispheric effects did not survive the correction for multiple comparisons  . Similar as in MCI-CB, effects of VP volume reductions in MCI-CC  did not survive the correction for multiple testing. Significance and p-values were similar when using raw volumes instead of volumes relative to eTIV. However, two right hemispheric effects in hippocampal subfield volumes did not quite achieve the level of significance   in dorsal aspects of the pulvinar, VP and lateral posterior nuclei as well. Thalamic expansions in turn were limited to ventral and medial parts. MCI-CB revealed expansions in bilateral ventral aspects of the central nuclei, VA, VL, and VP. MCI-CC revealed the same, though more pronounced pattern of expansions in both hemispheres, with additional expansion in the medial aspect of the medial dorsal nuclei.Thalamic contractions and expansions are presented in Figure Striatal displacements are presented in Figure t-values constituted a local minimum (or maximum), the average of the displacement values in mm was computed for each subject. This was done separately for each structure and hemisphere. The analyses revealed contractions of the right thalamus and left striatum having an AUC of 0.98  and of 0.96  for discriminating HC from MCI-CB  and increased accuracy and statistical significance when compared to the right hippocampus volume . The same AUC analyses carried out for thalamic expansions , thalamic contractions left  and striatal expansions left  revealed lower but still statistically significant accuracy for discriminating HC from MCI-CB.For these analyses, between group t statistics from comparison between HC and MCI-CB were used. More precisely, for all vertices whose In the current study, we used a combination of novel  and well-established  structural imaging techniques to characterize the neuroanatomy of MCI-to-AD converters. We examined the neuroanatomy in the MCI-to-AD converters using data acquired both at baseline and time of conversion. In addition, we compared baseline data from stable MCI subjects, comparing them with data from group-wise matched HC in a cross-sectional manner.There was no significant difference in cortical thickness in MCI-S compared to HC, but in MCI-CB in left parahippocampal regions including the uncus with further propagation to bilateral regions in MCI-CC. This pattern is consistent with the literature , control subjects were recruited for group-wise matching to each patient group with respect to these potentially confounding factors.Additionally, the comparison of the data between MCI-S and MCI-CB, and between MCI-CB and MCI-CC would be of high interest. However, and apart from the cross-sectional nature of the present study, these analyses require accounting for confounding variables , and increase the number of comparisons. Generally speaking, and in consideration of the rather low sample sizes in the present study, additional analyses would further reduce the power of the present results. Similarly, ROC analyses discriminating MCI-CB from MCI-SB based on striatal and thalamic shape alterations is relevant. However, and for similar reasons and consistency, the main aim of the ROC analyses in the present study was to demonstrate similar if not improved abilities of striatal / thalamic shape alterations compared to hippocampal volumes for AD to discriminate between MCI-CB from HC. But first and foremost, the aim of the present study was to investigate morphometric differences between patient and well matched control groups to establish the sophisticated analysis techniques on images obtained from a 1.5 Tesla scanner. Nevertheless, further studies are now required to confirm our results in larger samples allowing for additional and more advanced statistical models.n = 3) and amnestic MCI multiple domain subjects (n = 7). More precisely, both MCI groups showed memory impairment and impairment in a language-related function (confrontation naming) as well as in semantic memory and executive functioning . Interestingly, however, MCI-S but not MCI-CB showed further impairments in additional executive tasks  as well as in attentional abilities (TMT A and TMT B). These attentional deficits in MCI-S might be at the basis of the other cognitive deficits in this group, resulting in a more widespread and global pattern of cognitive impairment when compared with MCI-CB–with remaining abilities for everyday life at the same time. Generally speaking, these findings might represent non-typical-AD pathology rather than early stage AD in the present MCI-S group, and this assumption is further supported by the morphometric findings in this group . However, the AD typical cognitive impairment, the pattern of cortical thinning and hippocampal atrophy in MCI-CB and MCI-CC, together with a cognitive profile uncharacteristic for AD and the absence of morphometric alterations in MCI-S, is supportive of AD pathology in MCI-CB and MCI-CC. Additional markers of neurodegeneration such as CSF levels of phosphorylated Tau together with amyloid imaging may provide additional information on this matter in future longitudinal studies. Also, vascular risk factors such as hypertension, diabetes mellitus, hyperlipidemia, etc. represent main risk factors for the development of dementia. This information, however, was not available for all subjects and could therefore not be added in the statistical models.MCI-S has been described as a potential early stage of AD , and by an International Short Visits Grant from the SNF to SL (IZK0Z3_151124). MC is funded by the Canadian Institutes of Health Research, National Sciences and Engineering Research Council of Cananda, Weston Brain Institute, Alzheimer's Society, and the Michael J. Fox Foundation for Parkinson's Research. RN and CH are founder/co-founder and board members of Neurimmune Holding AG, Schlieren.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. RN and CH are founder/co-founder and board members of Neurimmune Holding AG, Schlieren.