Magnetic resonance-guided laser interstitial thermal therapy (MR-gLiTT) is a novel minimally invasive treatment approach for drug-resistant focal epilepsy and brain tumors. Using thermal ablation induced by a laser diode implanted intracranially in a stereotactic manner, the technique is highly effective and safe, reducing the risk associated with more traditional open surgical approaches that could lead to increased neurological morbidity. Indications for MR-gLiTT in pediatric epilepsy surgery include hypothalamic hamartoma, tuberous sclerosis complex, cavernoma-related epilepsy, SEEG-guided seizure onset zone ablation, corpus callosotomy, periventricular nodular heterotopia, mesial temporal lobe epilepsy, and insular epilepsy. We review the available literature on the topic and present our series of patients with drug-resistant epilepsy treated by MR-gLiTT. Our experience, represented by six cases of hypothalamic hamartomas, one case of tuberous sclerosis, and one case of dysembryoplastic neuroepithelial tumor, helps to confirm that MR-gLiTT is a highly safe and effective procedure for several epilepsy conditions in children. Magnetic resonance-guided laser interstitial thermal therapy (MR-gLiTT) is a novel and very promising minimally invasive therapeutic approach in the field of neurosurgery. The two major indications for this type of neurosurgical treatment are drug-resistant focal epilepsy and brain tumors . LiTT usThere are two LiTT systems available on the market: the Visualase system  and the NeuroBlate system , the latter only marketed in North America so far. Both systems use color-coded thermal maps superimposed on MR and have a software that, using the Arrhenius equation that estimates cell death based on the temperature and time dependence of protein denaturation processes, represents areas of irreversible damage . The estMRgLiTT has been licensed for use in Europe in spring 2018.In this paper, we report on the state of the art on the use of MRgLiTT in pediatric epilepsy surgery. Moreover, we present the case series of patients with focal epilepsy associated with lesions treated by MRgLiTT at Giannina Gaslini Children's Hospital in Genoa .Hypothalamic hamartomas (HHs) are rare, tumor-like, and non-progressive malformative lesions that occur during fetal development from ventral hypothalamus . AlthougComplications with LiTT are often transient but can be severe. In their recent series of 18 patients, Xu et al. . Other aTSC is a neurocutaneous syndrome that variably involves the brain, skin, kidney, heart, and lungs. Epilepsy is the most common clinical manifestation of TSC, occurring in approximately 90% of cases . EpileptCavernomas are mulberry-like vascular malformations often found in brain and spinal cord. Brain cavernomas can determine irritation (epilepsy) or deficiency symptoms . LiTT haThe SEEG is a method of Functional and Stereotactic Neurosurgery that allows an invasive EEG study in order to identify the seizure onset zone (SOZ) in cases where non-invasive diagnostic studies have not produced good anatomo-electro-clinical correlations . More reHuang et al. recently published a retrospective study of a case series of six patients (three children and three adults) who underwent callosotomy using LiTT. Engel outcomes for completion corpus callosotomy by LiTT were similar to reported outcomes of open completion callosotomy, with seizure reduction primarily observed in adult patients . As withPeriventricular nodular heterotopias (PNHs) are malformations of cortical development characterized by disorganized but histologically normal aggregates of neuronal and glial cells. They are often associated with drug-resistant epilepsy . The anaTreatment of mesial temporal lobe epilepsy (MTLE) by means of LiTT has been addressed by some studies concerning the adult population , 32. It Insular epilepsy is another potentially interesting area of application of LiTT in pediatric epilepsy surgery . The insFocal cortical dysplasias and dysembryoplastic neuroepithelial tumors (DNETs) are the most frequent causes of drug-resistant epilepsy in children . NeverthThe use of MR-gLiTT in Epilepsy Surgery requires the placement, through a micro-hole drill, of a laser fiber within an intracranial lesion target using stereotactic methods . A skull anchoring system is mounted on a steel rod and is screwed into the microhole at the calculated angle, forming a solid anchor point for insertion of the laser fiber. The laser probe is then positioned through the above system until it reaches the intracranial target. Heat delivery is monitored in near real time by MR thermography, as explained above, until the desired ablation is achieved; multiple heat deliveries may be required during the single procedure, and repositioning of the laser probe may be necessary .Patient demographics, preoperative seizure frequency, seizure semiology, and postoperative outcomes are listed in Overall, the age of the patients ranged from 11 months to 15 years. The mean age at surgery was 6 years (73 months). Six of eight (75%) patients were male. Pathologies were represented by HH in six patients, TSC in one case, and DNET  in another one. Precocious puberty was present in three patients with HH ; two of them received pharmacological treatment before surgical intervention.Patients were studied with prolonged video-EEG monitoring and brain MRI.Preoperative seizure frequencies ranged from 1 event per month to more than 40 per day. Seizure types were gelastic and dacrystic seizure in the six HH patients, five of them presented with additional seizure types . In patient nr 7 and 8, we could not assess the level of awareness impairment during the seizure . Patients affected with DNET and TSC presented with focal seizures with preserved awareness.In the HH cases, the EEG data, together with anatomical localization of the hamartoma, were used to decide the side of the entry point of the laser fiber on the skull.The trajectories of the laser fiber were designed using the following principles. For the HH cases, the target point of the laser probe was placed at a point between the lateral two-thirds and the mesial third of the maximum diameter of the lesion, measured on coronal and axial planes. Then, the intralesional target point was shifted slightly in a caudal and dorsal direction in the sagittal plane to maximize heat dissipation by the “heat sinks,” represented by the basal arachnoid cisterns and blood vessels. The trajectories of the laser probe were planned in order to avoid structures that could potentially be damaged mechanically or by the heat developed by the laser, such as cerebral blood vessels, optic tracts and/or optic chiasm, mammillothalamic tracts, and fornices. The laser system used  made it possible to monitor in almost real time the temperature variations reached at certain points, in order to avoid damage to the perilesional nervous structures, automatically stopping laser delivery in the event of an increase in temperature beyond a previously set level  were completely seizure-free after the procedure.One patient (HH) experienced two focal episodes in the 2 months after the procedure; to date, he is seizure-free with only one anti-seizure medication (ASM).Two patients with HH experienced rare, non-disabling seizures after the procedure (classified as Engel Ib). One patient with HH (#7) showed no seizure improvement (it is worth noting that the patient presented with a large HH).In the TSC patient with drug-resistant epilepsy associated with multiple cortical tubers, a significant reduction in seizure frequency (from two to three episodes per day to <1 episode per week) was achieved, along with an improvement in his behavioral hyperactivity.Postoperatively, no patients experienced new neurological morbidity or endocrine dysfunction, with two of them experiencing acute postoperative seizures (APOS). The mean hospital stay was 6 days, and 100% of patients regained normal preoperative motility and activity on the second day after the procedure. In three patients, neurodevelopmental assessment  before and after the surgical procedure  showed improvement in cognitive and social behavior.Although follow-up is overall very short (in two cases is shorter than 3 months) and the number of patients is still quite small, our data are globally consistent with those reported in literature, showing that the high majority of HH patients become seizure free after the procedure , 17. OnlIn our opinion, besides from seizure outcome, the most striking aspect of the procedure is the regain of habitual motility and functioning in such a short time compared to traditional surgery, with a reduced hospital stay and convalescence time .The relevance of the technique is also fundamental in treatment of multiple tubers as in TSC cases: such a minimally invasive procedure could potentially make it possible to perform multiple surgeries on multiple tubers, something that is not advisable with a classical open technique.Although the evidence on the therapeutic results of LiTT in the pediatric setting is still limited in terms of both quantity and scientific quality, there is no doubt that, at least in perspective, it may represent a first line of minimally invasive treatment of diseases associated with drug-resistant epilepsy. This consideration is particularly pertinent for those brain lesions difficult to access with the methods of traditional surgery. The overall complication rate is considered more than acceptable compared to traditional surgical techniques, the epileptological outcome obtained with LiTT is commonly evaluated as good. It remains to be clarified what will be the real economic costs of this innovative technique on the various health systems. Carefully designed scientific studies will in any case have to take into account not only the currently high costs of this new technology but also the indirect cost savings through a shorter duration of hospitalizations, cost savings on ASMs, and medium- and long-term follow-up. Ultimately, although future prospective, multicenter studies will better define the role of LiTT in neurosurgery, it is more than reasonable to believe that, with increasing ease of use and a more robust demonstration of efficacy, LiTT will rapidly become an extremely attractive therapeutic method for the treatment of many conditions associated with drug-resistant epilepsy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participant's legal guardian/next of kin.AC: ideation, data collection and analysis, writing and critical revision of the paper. EC, MP, and DT: data collection and critical revision of the paper. MM, GD, and GP: critical revision of the paper. LN: data analysis and critical revision of the paper. All authors contributed to the article and approved the submitted version.DINOGMI contributed to this work within the framework of the DINOGMI Department of Excellence MIUR 2018-2022 (legge 232 del 2016).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nanowire structures with high-density interfaces are considered to have higher radiation damage resistance properties compared to conventional bulk structures. In the present work, molecular dynamics (MD) is conducted to investigate the irradiation effects and mechanical response changes of GaAs nanowires (NWs) under heavy-ion irradiation. For this simulation, single-ion damage and high-dose ion injection are used to reveal defect generation and accumulation mechanisms. The presence of surface effects gives an advantage to defects in rapid accumulation but is also the main cause of dynamic annihilation of the surface. Overall, the defects exhibit a particular mechanism of rapid accumulation to saturation. Moreover, for the structural transformation of irradiated GaAs NWs, amorphization is the main mode. The main damage mechanism of NWs is sputtering, which also leads to erosion refinement at high doses. The high flux ions lead to a softening of the mechanical properties, which can be reflected by a reduction in yield strength and Young’s modulus. Compared with traditional bulk materials, semiconductor nanowires (NWs) on the one-dimensional nanoscale have excellent mechanical and optoelectronic properties ,2 due to+ irradiation experiments on GaAs/AlGaAs core-shell NWs at room temperature and found that the minority carrier lifetime is closely related to the irradiation-induced defect density by photoluminescence (PL) method. In addition, the size dependence of the carrier lifetime damage factor of GaAs NWs is mainly attributed to a special dynamic mechanism of defect annihilation, as summarized by them  direction to calculate the stress-strain curves of un-irradiated and irradiated GaAs NWs to evaluate the mechanical properties under the irradiation effect. Here, five state points with the number of injected ions of 0, 50, 100, 150 and 200 are taken. The stress-strain curves are shown in As discussed previously, defects are the main cause of the variation in the mechanical properties of irradiated GaAs nanowires. However, in this setting, the effects of different types of defects, such as internal and surface defects, are different due to the special structural nature of the high-density interfaces of nanowires . In thisIn this paper, we use MD simulations to reveal the defect generation, accumulation mechanism and mechanical response changes of GaAs NWs under heavy-ion irradiation. In terms of defect generation, sputtering damage of NWs is the main damage. What is more, the presence of surface effects at lower energies makes the number of nanowire defects larger than that of bulk structures. The low stopping power of the nanowire structure for high-energy particles means that less collisional energy transfer occurs, which is the main reason for the non-linear relationship of the defects. In terms of defect accumulation, defects exhibit a special mechanism of rapid accumulation to saturation, which is closely related to the surface dynamic annihilation process. Under the continuous ion injection, GaAs nanowire structure transformation mode is mainly an amorphous pathway, when the nanowire damage region shows erosion refinement phenomenon. In addition, there is also a small amount of ZB atoms transformed into WZ structures. The high flux ions lead to a softening of the mechanical properties, which can be reflected by a reduction in yield strength and Young’s modulus. Among them, for yield strength, internal defects have a greater impact than surface defects, but for Young’s modulus, this situation is reversed.


Neurofinance investigates the brain's activity in financial agents' decision-making processes. The most salient stages of the decision-making process are the collection of visual and auditory stimuli while receiving information on investment options, the selection and classification of such stimuli, their processing, and the interpretation. These phases are influenced by genetic and personality traits, implicit (unaware) memory of experiences, and by risk, or uncertainty, or ambiguity perception related to the available financial information and the market context. The brain automatically and unconsciously does many things before a decision is made and agents are aware of it. Neurofinance combines financial theories with psychology and neuroscientific insights to explain, in an interdisciplinary way, the widespread irrationality in the behaviour of financial operators. The role of emotion, mental status, biases, stress, personality, gender, age, and experience is therefore detected and analysed using different non-invasive clinical tools . Neurofinance researchers collect neural signals to understand what happens in the black box during the financial decision-making process. Since 2005, the articles and scientific texts in Neurofinance have increased annually. In this Neurofinance Research Topic, the first type of article includes the works of: Ceravolo et al.Attention Allocation to Financial Information: The Role of Colour and Impulsivity Personality Trait. The authors use eye-tracking technology, with an ecological protocol, to examine the patterns of eye movements in the early phases of information acquisition, during the reading of the KIID , disentangling the independent role of colour and impulsivity at modulating attention distribution. They observed that the increased attention induced by colour compensates for individual impulsivity, providing relevant insights for regulators who aim at increasing transparency and investors' protection. Jin et al., in their article ‘Stimulating the Dorsolateral PreFrontal Cortex (DLPFC) decreases the Asset Bubbles’ discuss the causal relationship and transmission mechanism of the DLPFC and asset bubble and examine different forecasting rules across individuals. The authors inferred that cognitive ability might be an important transmission mechanism in reducing asset bubbles. Wang et al., in their article ‘Anodal tDCS over the right temporoparietal junction lowers overbidding in contests’, using tDCS to modulate the activation of the rTPJ, investigate the neural underpinnings of overbidding—that is a commonly observed behaviour in competitive social interactions such as contests or auctions—and how the rTPJ impacts bidding behaviour. Findings suggest that the activation of the rTPJ in contests affects overbidding and bidding strategy, and further confirm that, in a competition context, the rTPJ is involved in the inference of mental states. In the second type of article, we consider the works of: Liao et al.Exogenous Testosterone increases Decoy Effect in Healthy Males. The authors studied the role played by testosterone in financial decisions and how it modulates decision-making when an asymmetrically dominated decoy option is introduced in a choice set. Results showed that participants in the testosterone group made fewer consistent choices and more target choices  than participants in the placebo group. These findings are interpreted in light of the dual-process theory based on existing evidence suggesting that testosterone promotes more intuitive and automatic judgments in human decision-making. Ramchandran et al., in their article ‘The Role of Emotional vs. Cognitive Intelligence in Economic Decision-Making Amongst Older Adults’ explore the links between emotions, bio-regulatory processes, and economic decision-making, which are well established in the context of age-related changes in fluid, real-time, decision competency. They explored this decision-making competency in the neurobiology of ageing by examining the neuroanatomical correlates of intelligence and decision-making, especially in regions that form a subset of the human mirror-neuron system. The data indicate that emotional intelligence plays a significant role in the economic decisions of older adults. McCormick et al., in their review article ‘Neural Underpinning of Financial Decision Bias in Older Adults: Putative Theoretical Models and a Way to Reconcile them’ focus on the topic that older adults face a perspective of reducing their economic wellbeing, autonomy, and happiness. Some factors such as increased medical expenses, reduced mobility, deficits in eyesight and hearing, and others lead them to retire, accepting a decreasing retirement income. They describe the processes of PFC decline, altered E-RP, and altered DMN in ageing, which, together, can affect financial decision-making. An alternative framework for understanding the neural network underpinnings of financial decision bias in older adults is proposed. Extracting event-related potentials (ERPs) from electroencephalographic data of a sample of top executives in real enterprises, Zang et al., in their paper ‘The Neural Basis of Herding: Decision in Enterprise Clustering: An Event-relatEd Potential Study’ examined the neural basis and processing of herding decisions in people's daily lives, showing the existence of herding tendency in business decisions. ERP results indicated that anti-conformity choices induced a larger amplitude than herding choices, demonstrating that participants might experience larger perceived risk and more decision conflict when they processed anti-conformity choices. In their fMRI study, ‘Cash, Card, Smartphone: The Neural Correlates of Payment Methods’, Ceravolo et al. focus on information technology innovations that have pushed towards the irreversible digitalization of payments. The authors tried to understand if and how brain activity can be modulated by the method of payment , the paid amount, and/or both. They observe a greater activity of areas processing the perceived utility of motor behaviour , and the individual emotional involvement . The authors suggest that cash enhances the salience and negative affective valence of paying by cash. The future development of Neurofinance will not only concern how to improve knowledge of the neural aspects of a financial agent's decision-making, but also new research areas related to building bridges between neuroscience and applications in business, to increase the value added, transparency, particularly in the financial market, and higher effectiveness of financial investor protection rules.Neuroeconomics has recently emerged as a new interdisciplinary scientific area aimed at examining the role of the brain that, as a physiological organ, influences, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plants benefit extensively from endophytic bacteria, which live in host plant tissues exerting no harmful effects. Bacterial endophytes promote the growth of host plants and enhance their resistance toward various pathogens and environmental stresses. They can also regulate the synthesis of secondary metabolites with significant medicinal properties and produce various biological effects. This review summarizes recent studies on the relationships between bacterial endophytes and medicinal plants. Endophytic bacteria have numerous applications in agriculture, medicine, and other industries: improving plant growth, promoting resistance toward both biotic and abiotic stresses, and producing metabolites with medicinal potential. Their distribution and population structure are affected by their host plant’s genetic characteristics and health and by the ecology of the surrounding environment. Understanding bacterial endophytes can help us use them more effectively and apply them to medicinal plants to improve yield and quality. The quality and yield of medicinal plants are significantly influenced by environmental factors, such as temperature, illumination, moisture, soil conditions, and the presence and identity of soil fauna . It is iBacterial endophytes are present in the flowers, leaves, roots, seeds, and stems of plants . BacteriThe community of bacterial endophytes is influenced by both biotic and abiotic factors, which shape their species composition, community structure, diversity, and functions . EnvironHere, we summarize the external factors that affect endophytic bacteria, and we survey the potential uses of them in growth promotion, pathogen resistance, and secondary metabolism of medicinal plants. Understanding and using these symbiotic relationships can enable us to more effectively cultivate valuable plants for human use and improve the quality and yield of medicinal materials.Morus L.) in the spring, whereas only Proteobacteria were found in the fall. The spring endophytic bacteria were also characterized by greater diversity and a larger number of species  compared with autumn endophytes   . Another content . Xu et a content . BacteriHieracium pilosella than on Gaultheria mucronata  Hell and Echinacea purpurea (L.) Moench, which demonstrated that these two medicinal plants and the respective compartments possessed different types of bacterial communities, suggesting the strong selective pressure and the low-degree strain sharing in the plant tissues   .Agrobacterium tumefaciens bacterial community may constitute an important component of their A. tumefaciens resistance  the development of innovative approaches for the separation and cultivation of endophytic bacteria in order to build a functional library of endophytic bacteria and investigate the effects of unculturable endophytes on medicinal plants; (2) studies on the effects of endophytic bacterial communities on medicinal plants; (3) artificial transformation of functional bacteria to give them additional beneficial functions; (4) strategies to establish symbiotic endophyte-host plant associations, and simulation of the symbiotic vs. parasitic relationships between endophytic bacteria and medicinal plants; (5) transmission mode , and (6) explorations of the mechanisms by which Dao-di medicinal materials are formed, with an emphasis on the role of endophytic bacterial community structure.Plants have evolved through continuous interaction with microbes, and it is evident that endophytic bacteria play significant roles in improving plant survival and adaptation . This reLQ and BZ reviewed and finalized manuscript. WW and WC completed the article writing. JW, SL, and YZ integrated information of tables, analyzed, data, and made pictures. All authors reviewed and approved 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.


Over the last several decades, colorectal cancer (CRC) has been one of the most prevalent cancers. While significant progress has been made in both diagnostic screening and therapeutic approaches, a large knowledge gap still remains regarding the early identification and treatment of CRC. Specifically, identification of CRC biomarkers that can help with the creation of targeted therapies as well as increasing the ability for clinicians to predict the biological response of a patient to therapeutics, is of particular importance. This review provides an overview of CRC and its progression stages, as well as the basic types of CRC biomarkers. We then lay out the synopsis of signaling pathways related to CRC, and further highlight the pivotal and multifaceted role of nuclear factor (NF) κB signaling in CRC. Particularly, we bring forth knowledge regarding the tumor microenvironment (TME) in CRC, and its complex interaction with cancer cells. We also provide examples of NF-κB signaling-related CRC biomarkers, and ongoing efforts made at targeting NF-κB signaling in CRC treatment. We conclude and anticipate that with more emerging novel regulators of the NF-κB pathway being discovered, together with their in-depth characterization and the integration of large groups of genomic, transcriptomic and proteomic data, the day of successful development of more ideal NF-κB inhibitors is fast approaching. From the Centers for Disease Control and Prevention (CDC), colorectal cancer (CRC), also known as bowel cancer, refers to cancers that originate in the colon or rectum. This is further defined by the positioning of the cancer, dictating whether it is termed colon cancer or rectum cancer.Decades ago, colorectal cancer was rarely diagnosed. However, now it is one of the leading carcinomas found in western countries and the fourth most lethal cancer worldwide ,2,3. CRCstage 0, aberrant cells grow up in the innermost layer (mucosa) and form tubercules in the colon or rectum, but these cells are non-cancerous )EQon Pharmaceuticals, and other drugmakers such as GlaxoSmithKline (GSK) and Johnson & Johnson, have been developing PRMT5 inhibitors to treat cancers. So far, a small number of candidate compounds have entered the early stage of clinical trials.In sum, this evidence affirms the inhibition of the NF-κB pathway as a promising therapeutic approach for CRC. Considering the important role of NF-κB, which is involved in many physiological processes, approaches that can avoid systemic NF-κB inhibition are more desirable. The development of nanotechnology for drug delivery has helped the use of minimal dosages of drugs, targeted to limited areas of the body . Though We are confident that, with more emerging novel regulators of the NF-κB pathway being discovered, together with the in-depth characterization of these newly identified biomarkers of CRC diagnosis and therapeutic response, along with the integration of large groups of genomic, transcriptomic and proteomic data, the goal of successful development of the most ideal NF-κB inhibitors will ultimately be achieved in the near future.


We believe that the established nanofabrication process will be very useful for expanding fundamental research and in further improving the applications of nanofluidic devices.Nanofluidics is supposed to take advantage of a variety of new physical phenomena and unusual effects at nanoscales typically below 100 nm. However, the current chip-based nanofluidic applications are mostly based on the use of nanochannels with linewidths above 100 nm, due to the restricted ability of the efficient fabrication of nanochannels with narrow linewidths in glass substrates. In this study, we established the fabrication of nanofluidic structures in glass substrates with narrow linewidths of several tens of nanometers by optimizing a nanofabrication process composed of electron-beam lithography and plasma dry etching. Using the optimized process, we achieved the efficient fabrication of fine glass nanochannels with sub-40 nm linewidths, uniform lateral features, and smooth morphologies, in an accurate and precise way. Furthermore, the use of the process allowed the integration of similar or dissimilar material-based ultrasmall nanocomponents in the ultranarrow nanochannels, including arrays of pockets with volumes as less as 42 zeptoliters (zL, 10 Nanofluidics involves the study of fluids at nanometer dimensions ,3,4,5,6.Nanochannels are the core components of most nanofluidic devices. The methods for fabrication of nanochannels vary depending on the substrate material. While several kinds of substrate materials such as glass, polydimethylsiloxane (PDMS), and plastics are used in the fabrication of microfluidic devices, the widely successful congener of the nanofluidic devices, glass, and particularly fused-silica (amorphous form of silica), is currently the sole substrate material that is ideal for the fabrication of nanofluidic chips. While silicon was initially used for fabrication of nanochannels by the direct use of well-established nanofabrication technologies for microelectronics , its optSeveral tools have enabled the fabrication of nanochannels in glass substrates. Standard photolithography incorporated with short-time wet or dry etching allows the fabrication of fused silica nanochannels with micrometer-scale widths and nanometer-scale depths, which are, respectively, defined by the practical resolution limits of photolithography and etching duration. Such nanochannels with large widths are also called planar nanochannels or one-dimensional (1D) nanochannels and have been employed in early studies for DNA extension . In contIn this study, we achieved efficient, accurate, and precise-fabrication of fine ultranarrow nanochannels with feature sizes of several tens of nanometers by focusing on two key processing parameters, the EB resist thickness and development time, among the various complicated nanofabrication processing parameters of the EBL coupled with dry etching processes. The use of an optimized nanofabrication process makes it possible to fabricate fine ultranarrow nanochannels and nanochannels with similar or dissimilar material-based ultrasmall nanocomponents. The fabrication process established in this study will aid the advancement of fundamental research and the further improvement of nanofluidic device-based applications.Nanofabrication was performed by combining nanopattern transfer with subtractive techniques (such as EBL and plasma dry etching) and/or processes of nanopattern transfer with additive techniques . These individual processes are briefly described herein, and further details are available in the literature ,34,35,36An EBL process was used for transferring nanopatterns, generated via computer-aided-design (CAD), onto a substrate. First, an EB resist  was spin-coated on a perfectly cleaned fused-silica glass substrate  using a spin coater . To investigate the effect of the thickness of the EB resist on nanofabrication, different spin-coating speeds of 2500, 3000, 4000, 4300, and 4500 rpm were applied to obtain EB resist layers with different thicknesses, which were further measured using a stylus surface profiler . Next, an electron beam under a standard beam condition was irradiated onto the EB resist-coated glass substrate using an electron beam lithography system . Finally, the EB irradiated-substrate was developed in xylene  in a thermostatic bath  at 25.0 ± 0.1 °C, and the resulting designed nanopattern was transferred to the EB resist layer.To transfer the EB resist nanopattern onto the glass substrate, a plasma dry etching process was applied using a reactive ion etching (RIE) system . In the etching process, fluorine gas  was used as the working gas to achieve an etching rate of 22–26 nm/min. After etching, the remaining resist was removed using a mixture  of dimethyl sulfoxide  and xylene.−5 Pa. Here, the thin Cr layer played a role in increasing the adhesion between the gold layer and the glass substrate. After the process for removing the Au/Cr on the resist using a mixture of dimethyl sulfoxide and xylene , the remaining Au/Cr parts formed gold nanopatterns on the substrate .Physical vapor deposition processes and lift-off were used to fabricate the gold nanopatterns. After transferring the computer-aided nanopattern designs to the resist layer on the substrate using the standard EBL process, 5 nm-thick chromium  and 30 nm-thick Au  films were sequentially deposited on the substrate using vacuum evaporation equipment  at 10Two types of 50 parallel 2D nanochannel structures  with and without pockets  were fabricated using the above-mentioned processes of EBL and plasma dry etching. This is illustrated in Gold nanogap structures  were fabricated using the above-mentioned processes of EBL, physical vapor deposition, and lift-off, and this is illustrated in The nano-in-nano structures were fabricated using a multiple-step EBL process  in ESI sMicrochannels were dry-etched on a fused-silica glass substrate after photolithography and the resulting inlet and outlet holes were penetrated using a diamond-coated drill . The nanEBL and the ensuing plasma dry etching processes involve several intricate parameters, such as the resist layer thickness, dose time, voltage and beam current of the electron beam, field size and number of dots, development time, plasma density, etching rate, etching selectivity, etching time, etc. All of these parameters are involved in the nanofabrication process; however, in this study, we focused on the resist layer thickness and development time, considering the required multiple processes in chip-based nanofabrication, which is different from the general processes for each individual nanopattern transfer steps, as described below .The chip-based nanofabrication process involves two transfer steps . The firTo determine the optimal resist thickness, the selection ratio of the resist to glass during plasma dry etching (second transfer) needs to be examined. In this study, the EB resist solution was diluted three-fold to achieve a sufficiently thin EB resist layer thickness. As revealed by preliminary experiments, the resist-to-glass selection ratio of etching (defined as the ratio between the etching rate of resist and the etching rate of glass) was approximately 1/1 for the case using three-fold diluted resist. Considering that our target depth of nanochannels was 35–40 nm and the thickness of the resist layer may not be uniform in the range of tens of nanometers, we determined the target EB resist thickness to be 70–80 nm to sufficiently prevent the glass area without nanochannels from being etched, according to our experience.However, to fabricate chip-based structures with feature sizes of tens of nanometers, investigation of the EB resist thickness is not sufficient. The development time, which is the time required to remove the EB-irradiated parts of the resist by dissolving in xylene, must also be investigated. The development time significantly affects the size of the EB resist patterns that form the nanochannels in the next transfer, and the optimal time depending on the EB resist thickness should be considered . SignifiTo investigate the influence of development time, the fabrication of small gold nanogaps at different conditions of different development times were studied . The golThe results of the optimization of the development time on the gold nanogap fabrication are shown in ® , to improve electrical conductivity to some extent for FE-SEM imaging. The fabrication of ultranarrow nanochannels with tens of nanometer linewidths were achieved by using the optimized conditions, with a resist layer thickness of 78 nm and a development time of 30 s. As a demonstration, the fabrication of ultranarrow nanochannels with a channel width of 40 nm as a target value was performed. To characterize the lateral line uniformity of the fabricated nanochannel, the width of each representative nanochannel a,b was mDue to the ultrahigh surface-to-volume ratios of nanochannels, surface morphology of the nanochannels dominate a variety of nanofluidic phenomena. Smooth nanochannel wall is usually favorable and desired for a variety of applications of nanofluidic devices. Hence, characterization of surface morphology of the fabricated nanochannels is important. Due to the ultra-narrowness of the fabricated nanochannels in this study, it is difficult to directly characterize the wall surface of the nanochannels by using AFM, which is a powerful tool to characterize surface morphology. This is because the micrometer sized cantilever of AFM is significantly larger than the width of the fabricated narrow nanochannels, making it difficult to measure the inner walls of the nanochannels. Thus, in this study, together with the narrow nanochannels, a wide microchannel  was simultaneously fabricated in the same glass substrate under the same conditions and was used for indirectly obtaining the morphological information of the fabricated nanochannels by using AFM. The results f,g reveaTherefore, the optimized fabrication process allows the fabrication of fine nanochannels with ultranarrow linewidths of several tens of nanometers, uniform lateral features, and smooth morphologies, in an accurate and precise way. In addition, considering the significantly shorter processing time of EBL coupled with dry etching used in this study  than that of the FIB milling process , the op−21 L)  with square  and rectangular  pockets with zeptoliter volumes  a. ® to improve electrical conductivity for FE-SEM imaging  of both types of pockets. Considering that the current studies using nanofluidic structures operate mainly with volumes at the femtoliter  to attoliter  levels, the use of such ultrasmall pockets hold potential for further extension of nanofluidics to the zL regimes in the future.The substrate after nanofabrication was also spin-coated with ESPACER imaging b. The deThe integration of functional  components of dissimilar materials in nanochannels would open new avenues in the fusion of nanofluidics with a variety of other fields. The fabrication of nanochannels integrated with ultrasmall dissimilar material-based nanocomponents was further demonstrated by incorporating the optimized fabrication process with the nano-in-nano integration technology previously developed by us ,34. The The fabrication of nanochannels integrated with gold nanogaps of desired distance was achieved b–e. AmonThe substrate comprising the nanochannels integrated with ultrasmall nanogap arrays was further bonded with another glass substrate comprising two microchannels to form the nanofluidic device g,h. In aIn this study, we established a process for the fabrication of ultranarrow nanochannels and nanochannels with ultrasmall nanocomponents in glass substrates by optimizing the nanofabrication process of the EBL coupled with dry etching. The thickness of the EB resist layer and development time were investigated in detail. The use of the optimized process allowed the efficient fabrication of fine glass nanochannels with sub-40-nm linewidths, uniform lateral features, and smooth morphologies in an accurate and precise way. The established process also enabled the integration of similar or dissimilar material-based ultrasmall nanocomponents in the ultranarrow nanochannels. The fabrication of such nanochannel structures is highly desirable for fundamental- and application-oriented studies in nanofluidics; however, this has remained a challenge. Therefore, we believe that the established fabrication process would prove exceedingly useful for expanding fundamental research and initiate further remarkable applications of nanofluidic devices.


The immune system's role is much more than merely recognizing self vs. non-self and involves maintaining homeostasis and integrity of the organism starting from early development to ensure proper organ function later in life. Unlike other systems, the central nervous system (CNS) is separated from the peripheral immune machinery that, for decades, has been envisioned almost entirely as detrimental to the nervous system. New research changes this view and shows that blood-borne immune cells (both adaptive and innate) can provide homeostatic support to the CNS via neuroimmune communication. Neurodegeneration is mostly viewed through the lens of the resident brain immune populations with little attention to peripheral circulation. For example, cognition declines with impairment of peripheral adaptive immunity but not with the removal of microglia. Therapeutic failures of agents targeting the neuroinflammation framework (inhibiting immune response), especially in neurodegenerative disorders, call for a reconsideration of immune response contributions. It is crucial to understand cross-talk between the CNS and the immune system in health and disease to decipher neurodestructive and neuroprotective immune mechanisms for more efficient therapeutic strategies. The immune system protects us from all possible threats that could endanger the homeostasis of the body. The primary two arms of this protective system are innate and adaptive immunity. Both are represented by specific cells that directly (cell-to-cell) or indirectly (via mediators) eliminate danger. The innate system is older phylogenetically as compared to adaptive, exists in most life forms, is rapid and non-specific, and is highly efficient right at birth. Adaptive immunity is present only in jawed vertebrates, matures over time, is specific, and creates memory cells that can protect the organism throughout life . Regardiin vivo imaging, and single-cell expression analysis revealed the existence of meningeal lymphatic vessels and opened new perceptions of the interaction between CNS and the immune system  integrity. All glial cells can be classified as immune-competent cells within the CNS and possess characteristics of innate immunity. They respond to neuronal injury with programs that include propagation, changes in morphology, production of inflammatory molecules, and debris clearing. The brain is separated from the rest of the body by the BBB, which blocks passage of cells including immune cells and many molecules including antibodies. In this respect, the brain received the status of an immune-privileged organ. First studies that led to this concept came from the 19th-century experiments with dyes that stained all tissues except the brain, suggesting that the brain is efficiently separated from the rest of the body. Discovery of the BBB as a shielded structure that keeps away peripheral immune cells and mediators, the absence of classical antigen-presenting cells in CNS, diminished expression of MHC molecules, and lack of the classic lymphatic system further strengthened the concept of the CNS as an immunologically barricaded organ . Howevere system , 11. Inde system . Moreovevice versa, they can play an important role in the diagnosis and as new therapeutics of CNS disorders .The involvement of immune mediators, especially cytokines and chemokines, in brain development starts right at the beginning and includes coordinating trafficking, proliferation, and differentiation of neuronal and glial cells.As early as day 19 after conception, human hematopoiesis starts to proceed within the yolk sac, creating erythromyeloid precursors for yolk sac macrophages, which further colonize the brain and become microglia cells. Microglia development is synchronized with neurogenesis. Neurogenesis happens under the supervision of the TGF-β cytokine superfamily, especially bone morphogenic proteins (BMPs), which repress the induction of the process. Additionally, in the embryonic brain, many gp130 family cytokines are present such as LIF, CNTF, CT-1, and NP, where they play a role in the neuroepithelial/radial glial cell (RGC) self-renewal . The rolIn the developing brain, microglia are involved in neurogenesis and synaptic pruning. Microglia actively eliminate and phagocytose neurons via the production of toxic products such as superoxide ions, nerve growth factor (NGF), and TNF, which induce apoptosis without inflammation triggered by CR3/DAP12 signal transduction . Microglin vivo as well as regulation of synaptic transmission is partially dependent on cholesterol also synthesized by astrocytes. Mice with interrupted lipid synthesis show lessened synaptic development and plasticity , and the production and response to cytokines. Of note, as innate-immune cells, microglia and astrocytes produce significant amounts of antimicrobial peptides (AMP) that provide immune surveillance against pathogens, regulate the immune response and inflammation within the CNS, and maintain a healthy tissue , 57. Croin vitro manipulations is recovered once microglia are implanted back into adult mouse brain , the most numerous glial cells, are star-like cells with long processes assembling a dense network with all other CNS cells. In the human brain, one astrocyte makes connections with ~2 million synapses  generated from radial cells in three waves in mice, and in humans around the embryonic–fetal transition . OPCs exnditions . The lacIn contrast to microglia cells, but similar to astrocytes, oligodendrocytes and OPCs belonging to the same cell lineage are commonly classified as non-immune glial cells. However, recent discoveries shed new light on the immune properties of oligodendrocyte lineage cells including the regulation of innate and adaptive responses. Reciprocally, both adaptive and innate immune cells can shape the fate of OPCs via a range of immune mediators and trophic factors . TogetheOligodendrocytes are the most susceptible cells of the CNS, and mainly due to oxidative damage and their own high metabolic speed and vast oxygen usage, they needed to produce a bulk of lipids . AdditioGlial cells as part of innate immunity are crucial players in a wide array of neurological disorders. Their primarily role in the brain is to protect the CNS from any insult and repair the nervous tissue after the injury.Astrocytes were found to respond to stimuli by changing morphology, upregulation of MHC and GFAP, and immune mediators' release. Astrocytes are in close contact with blood vessels and control and guard the infiltration of cells from the blood to CNS parenchyma . AstrocyMicroglia and non-parenchymal macrophages, after stimulation, change their morphology and resemble systemic macrophages, upregulate MHCs, and release cytokines and nitric oxide. Recent exciting discoveries based on new genetic, molecular, and pharmacological tools allow for a better understanding of microglia phenotypes during an injury or disease that can be detrimental or beneficial . InfectiGliosis is one of the characteristics of MS, and involvement of innate immunity is evident. Microglia respond to tissue damage by MHC class I, II, and co-stimulatory molecule upregulation, the release of cytokines and chemokines that further attract a plethora of cells like T cells, B cells, monocytes, and dendritic (DC)-like cells that add to the myelin sheath destruction by nitric oxide, and matrix metalloproteinases. In EAE, microglia react to injury through increased proliferation, directed migration, phagocytosis, activation of the NLRP3 inflammasome, and, consequently, the release of proinflammatory mediators . Both ti+ cells contribute to re-myelination  EAE –198.MS is an autoimmune disorder of the CNS caused by an aberrant, chronic progressive inflammatory response leading to demyelination and permanent neurological disability in young adults, especially women. The pathogenesis of MS possesses a strong immune component supported by the efficacy of selective and non-selective immunosuppressive therapies on the disease progression. However, the innate or adaptive immune system's contribution to the development and progression of the disease has not yet been fully elucidated. Immunological, genetic, and histopathological studies in mice and humans suggested a dominant involvement of peripheral adaptive immunity during the early phase of disease and innate immune reactions within the CNS during the progressive stage . AlthougBesides obvious inflammatory mechanisms in MS, this disease also has characteristics of neurodegeneration. Earlier, it was believed that neurodegeneration is secondary to inflammatory-demyelinating processes, but now, it is widely accepted that axonal loss proceeds in parallel with induction of acute white matter plaques . SeveralFor a long time, it has been established that CD4+ T cells play a major role in preventing spontaneous experimental autoimmune encephalomyelitis (Sp-EAE) , 212 as Accumulating evidence shows that in classic neurodegenerative diseases such as AD and ALS, besides phagocytes, a crucial role is played by the adaptive immune system, which contributes to disease progression and protection. Mice models of AD and ALS, deficient in T cells, display rapid disease progression , 219. SoRapid progress in high-resolution live image technologies, single-cell genomics, quantitative proteomics, and epigenetics brings discoveries in the field of neurology. The long-lasting dogma imposing separation of the peripheral immune system from the nervous system, linking these two only in extreme pathological conditions, becomes obsolete. Their co-existence and close interplay assure proper functioning and homeostasis of the CNS. Failure of clinical trials of CNS therapeutics (with the possible exception of MS), mostly immunosuppressive and anti-inflammatory, proves our poor understanding of immune sentinels' function in the CNS. It is time to reconsider the immune system as a close partner to the brain environment since it is crucial for brain development, homeostasis, and disease. Recent discoveries of meningeal lymphatic vessels changed our perception of cross-talk between CNS and the immune system. How meningeal immunity and reciprocal cross-talk between innate and adaptive immunity influence CNS health and disease is now of critical importance in neuroimmunology.AM wrote the first and final drafts, performed data mining, created figures, and contributed to editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nuclei detection is a fundamental task in the field of histopathology image analysis and remains challenging due to cellular heterogeneity. Recent studies explore convolutional neural networks to either isolate them with sophisticated boundaries (segmentation-based methods) or locate the centroids of the nuclei (counting-based approaches). Although these two methods have demonstrated superior success, their fully supervised training demands considerable and laborious pixel-wise annotations manually labeled by pathology experts. To alleviate such tedious effort and reduce the annotation cost, we propose a novel local integral regression network (LIRNet) that allows both fully and weakly supervised learning (FSL/WSL) frameworks for nuclei detection. Furthermore, the LIRNet can output an exquisite density map of nuclei, in which the localization of each nucleus is barely affected by the post-processing algorithms. The quantitative experimental results demonstrate that the FSL version of the LIRNet achieves a state-of-the-art performance compared to other counterparts. In addition, the WSL version has exhibited a competitive detection performance and an effortless data annotation that requires only 17.5% of the annotation effort. Along with the rapid development of deep learning and computer vision, histopathology image analysis has become a centrally important research area in the field of computational pathology. The density, morphology, and distribution of cell nuclei, in the microscopic images of tissue biopsy stained with hematoxylin and eosin (H&E) or immunohistochemistry (IHC), can provide quantitative support and significant clues for assessing both the cancer grades and prognosis ,2. NucleDeep learning approaches have been proven to produce encouraging results on histopathology images in various studies ,6,7. RecTo address the above-mentioned problems of counting-based approaches, a novel local integral regression network (LIRNet) for nuclei detection is proposed in this paper. Firstly, a rigorously designed loss function that restricts the local integral around each annotated point, instead of single pixel in the density map, is adopted in the proposed LIRNet. The local integral regression loss does not pre-define any hypothesis on the distribution of cell nuclei density, but still separates nuclei with clear background gaps. As a consequence, the LIRNet can both provide the evident localization of each nucleus and count the nuclei population. Secondly, since the local integral calculates the sum of the density values in a local area while ignoring the specific position of each annotated point, it is reasonable to extend the local area to artificially meshed image patches, which intuitively yields a weakly supervised learning (WSL) framework. In the WSL framework of LIRNet, the histopathology image is gridded into patches for nuclei detection. Meanwhile, pathologists only need to distinguish whether there is zero, one, or multiple nuclei in one image patch. As a result, instead of a pixel-wise centroid annotation, patch-level labels are exploited to guide the training of the WSL framework. Finally, the quantitative experimental results show that the fully supervised learning (FSL) version of the LIRNet achieves a state-of-the-art performance, and the weak annotation strategy greatly reduces the labeling cost by We propose a novel local integral regression network that allows both fully and weakly supervised learning frameworks for estimating the conspicuous location of each nucleus in histopathology images.We creatively design a patch-level annotation method to reduce the annotation cost and explore a weakly supervised learning approach for nuclei detection task.The comparative experimental results quantitatively show that the FSL version of the LIRNet achieves a state-of-the-art performance, while the WSL version has exhibited a competitive detection ability with much less annotation cost.In summary, the key contributions of this paper are as follows:The rest of this paper is organized as follows: Traditional nuclei detection methods rely on the extraction of hand-crafted features such as color, texture, edge, and intensity information ,19,20. TLearning-based nuclei detection can be achieved from two aspects: segmentation and counting. The segmentation-based approaches aim to predict a pixel-wise probability map of the histopathology image, in which each nucleus is supposed to be separated by clear boundaries. However, the counting-based algorithms intend to estimate a density map of cell nuclei where the local maxima indicates the predicted position of each nucleus.Segmentation: In 2017, Kumar et al.  adopted Counting: Most of the nuclei detection studies ,26,27,28Fully supervised object localization requires bounding box labels or point annotations, which suffers from costly manual annotation. Accordingly, many studies ,35,36,37MBM cells: To assess the contribution of each part of our approach, we adopt the Modified Bone Marrow (MBM) dataset [ dataset ,38 that CA cells: To evaluate the performance of our WSL algorithm and compare the proposed FSL framework with state-of-the-art counterparts, we also employ the colorectal adenocarcinoma (CA) dataset [ dataset  containiPSU cells: Additionally, a dataset [ dataset  from PenTwo different data annotation strategies are applied to the proposed LIRNet for nuclei detection. On one hand, the fully supervised learning framework requires all of the point annotation centered at each nucleus a to yielConsidering that the nuclei are not uniformly distributed in most histopathology images (with high-density nuclei) which causes a label imbalance between The schematic diagram of the LIRNet architecture is shown in Besides, a nonlocal module  is introkth object area and the number of discrete object areas, respectively. Unlike the continuously distributed density map employed in traditional counting-based approaches ,13,27, tWhile in this work, we abandon the hyperparameter We find that the kth image patch As described in the data annotation and function :(5)Lk=taIn this work, we also replace each loss item mentioned above with the Equation  is rewriIt should be noted that there is no quations  and 6)  |·| operN local maxima on the density map, where N denotes the integral of the predicted density map, the nuclei locations can be obtained. A simplified version of non-maximum suppression (NMS) [r centered at each local maximum.Post-processing algorithms on the density map, which could be predicted by the the LIRNet, are desired to obtain the accurate location of an individual nucleus. According to the traditional experience of counting-based approaches ,13, the on (NMS)  is adoptP), recall (R), and F1 score , and 3rd quartile (Q3) of the distribution of the Euclidean distance between each detected nucleus and its nearest annotated center of nucleus.The object area for local integral is generated by a square kernel centered at an individual annotated point. The size of the square kernel is set according to the average distance between each nucleus and its 2 nearest neighbors (to reduce the influence of repeat labeling on the same nucleus) in the training dataset. As a result, the kernel size is set to 15, 11, and 13 pixels for the MBM, CA, and PSU datasets, respectively, which is exhibited in Equation  and the Equation  are set Data augmentations, including rotation and flipping, are conducted on the images to promote the accuracy of location. Besides, both the original image and its augmented form are simultaneously fed into the network at each training iteration, which is conceived to promote the detection performance. The training is optimized by the Adam optimizer  with a bWe first evaluate the performance of the FSL version of the proposed approach and compare it with state-of-the-art counterparts. Afterwards, the performance and the ability to reduce the annotation cost of the WSL framework are extensively evaluated on the CA and MBM datasets. Finally, we quantitatively assess the contribution of each part of our algorithm.We compare our LIRNet with eight competitive approaches on the CA cells dataset, including stacked sparse autoencoder (SSAE) , local iThe quantitative results of the comparative performance are shown in  ℓ2-tanh  and ℓ1 lr papers ,7,10,39.We also evaluate the proposed FSL framework on the PSU dataset , and the TSP-CNN . NeverthWe count and compare the cost of both the proposed patch-level labeling and the conventional point annotations, so as to measure the capability of our WSL framework on reducing the data annotation cost. It could be quantified by the number of mouse clicks. The experimental results on the CA dataset are illustrated in Applying the former annotation strategy, at most, 130 patch-level labels per image are provided on average, which reduces the annotation cost by at least d SC-CNN , and simThe WSL results on the MBM dataset are exhibited in To verify the ability of a nonlocal module on extracting global features of cell morphology and exploring the relationships among different positions, we first evaluate and compare the detection performance of different network variants. The experimental results on both the MBM and CA cells dataset are exhibited in In order to explore the effectiveness of the loss function design, we perform an ablation study between the local integral regression (LIR) loss (Equations  and 2)))2)) and Since our approach adopts the In the training stage, both the original image and its randomly augmented form (rotation/flipping) are simultaneously fed into the network at each iteration. To exhibit its contribution, we also compare the performance with and without employing this training strategy on the (We manually set each object area to the uniform square kernel according to the average distance between two nuclei in the histopathology images, as shown in In addition, we attempt to explain how the weakly supervised nuclei detection works in three aspects. First of all, the positions of the nuclei and the specific numbers remain unknown for the WSL training procedure. By comparing empty patches with single-nucleus ones, the LIRNet manages to distinguish and localize the nucleus from the background. Secondly, both single-nucleus and multi-nucleus patches provide weak counting supervision for the proposed network. Therefore, the LIRNet is capable of learning to count the number of nuclei, particularly for the regions with high-density nuclei. Thirdly, the nonlocal module in the LIRNet can effectively spread the learned capability of nuclei localization to the multi-nucleus patches on account of the similarity in appearance of nuclei with identical morphology.In this paper, we propose a novel local integral regression network that allows both fully and weakly supervised learning frameworks on nuclei detection. Compared with other fully supervised learning approaches, the FSL version of LIRNet achieves state-of-the-art detection performance, while the WSL version of LIRNet possesses a competitive detection performance and an effortless data annotation that requires much less annotation effort. Prospectively, the proposed approaches could offer a benefit to pathology practice in terms of a quantitative analysis of tissue images, and potentially lead to a better understanding of disease. Besides, considering the fact that LIRNet promotes the precision of location, it can conceivably be extended to other computer vision applications, for instance, the crowd detection issues.


The fabrication of porous metal structures usually involves complicated processes such as lithography or etching. In this study, a facile and clean method based on thermal evaporation at high pressure is introduced, by which a highly porous, black colored structure of Au can be formed through the control of homogeneous nucleation and growth during evaporation. The deposited films have different morphologies, from columnar to nanoporous structures, depending on the working pressure. These porous structures consist of Au nanoparticle aggregates, and a large number of nano-gaps are found among the nanoparticles. Thus, these structures indicate a much higher intensity of surface-enhanced Raman spectroscopy (SERS) when compared with commercial SERS substrates. The SERS intensity depends on the working pressure and thickness. Even circumstances that can induce agglomeration of nanoparticle aggregates do not deteriorate the sensitivity of SERS. These nanoporous structures based on high-pressure thermal evaporation are expected to provide a new platform for the development of low-cost and highly sensitive chemical sensors. Rapid and accurate probing of extremely small numbers of molecules has been a significant issue across several fields such as chemical sensors and pharmaceuticals. Recently, rapid and precise diagnostic tool kits have been developed for bacteria and viruses, and the COVID19 pandemic has boosted their significance. Surface-enhanced Raman spectroscopy (SERS) is one of the promising detection techniques because of its high sensitivity and simplicity of measurement ,5,6,7,8.SERS utilizes the plasmonic enhancement of the local electric field ) that occurs at nanoscale gaps of less than 10 nm, or “hot spots,” in various porous structures of gold or silver. These plasmonic nanostructures are known to amplify Raman signals by several orders of magnitude, which enables the probing of tiny amounts of target molecules ,15,16,17One typical approach is the dispersion of metal nanoparticles synthesized using wet chemical processes ,32,33,34Another approach is the cross-stacking of gold nanowire arrays through the nanotransfer printing method, as demonstrated by Jeong et al. . In thisTherefore, commercial SERS substrates are being produced by dry etching of Si to fabricate columnar structures with nanosized openings among the columns and consecutive surface coating with gold or silver to induce LSPR ,38,39. WHerein, we introduce another approach to obtain highly sensitive SERS substrates that is clean and can be applied to large-scale production. In this work, a nanoporous gold structure was successfully fabricated by simple high-pressure thermal evaporation. It requires neither a complicated dry-etching process nor the deposition of gold under high vacuum. The nanoporous gold developed in this work consists of gold nanoparticle aggregates, and the LSPR arising from the hot spots among the aggregates induces a much-enhanced SERS activity compared to the commercial SERS substrate. In addition, it endures harsh environmental conditions such as thermal annealing. Owing to the isotropic evaporation of the source material, large SERS substrates can be obtained from a small amount of gold.−3 Torr. After filling the chamber with Ar to adjust the pressure, the Au wire was heated and evaporation was initiated. When the evaporation was completed, the power supplied to the evaporation source was quickly removed to prevent coalescence of the nanoparticle aggregates because of the residual heat. A shield installed between the source and the wafer also blocked the radiation heat from the source during evaporation. The effects of various control parameters, such as working pressure, evaporation time, deposition temperature, and annealing, on the evolution of morphology were explored.Nanoporous Au structures were fabricated on chilled Si wafers using a high-pressure thermal evaporation process. A detailed description of this process has been reported in previous studies ,42. ContStructural characterization was performed using X-ray diffractometry , scanning electron microscopy , and transmission electron microscopy . The reflectance of the deposited nanostructure was measured using UV-Vis spectroscopy .For the surface-enhanced Raman spectroscopy measurement, a high-resolution Raman spectrometer  was utilized for rhodamine 6G (R6G) probe molecules diluted in de-ionized water. A 532-nm laser (0.5 mW) was irradiated, and the signal was accumulated for 10 s. Raman activity was measured for nanostructures with various surface morphologies, and the sensitivity was examined using different concentrations of R6G.During thermal evaporation under a relatively high pressure, the evaporated metal atoms encounter repeated collisions with each other owing to the gas molecules while traversing through the deposition chamber. These collisions induce the formation of large clusters of evaporated metal atoms, whose surface mobilities are suppressed owing to their sizes as they arrive at the substrate surface. Finally, these clusters adhere to similar clusters that arrived earlier and do not diffuse, forming porous structures. In contrast, as the working pressure decreases, the evaporated atoms arrive at the substrate without collisions. These adatoms have a higher potential of diffusing on the surface, and, therefore, a denser structure should be formed. As indicated by the SEM analysis exhibited in The highly porous Au thin film is represented by black color in the inset of The thickness of the nanostructured Au can be controlled by adjusting the evaporation time. As presented in As exhibited in −1 increased as the pressure increased to 10−1 Torr. However, it began to decrease below 10−1 Torr. This is because the structure formed at lower pressure has a reduced number of nanogaps, whereas the structure formed at higher pressure also has low LSPR owing to the enlarged nanoparticle size. It should be noted that the Raman intensity for the nanoporous Au formed at 1 Torr is expected to be increased by reducing its thickness because the enhancement of the Raman intensity is dependent on the thickness of the nanoporous Au, as indicated in −6 M for R6G molecules, as displayed in Nano-gaps formed among the Au nanoparticles enhanced the LSPR and increased the Raman intensity, as noted at the end of the preceding section. As indicated by the comparison presented in Compared to the commercial SERS substrate developed by Silmeco, which is fabricated by the dry etching of silicon wafer followed by the deposition of Au, the nanoporous Au investigated in this work exhibited much higher Raman intensity, as presented in Another peculiar feature of the high-pressure thermal evaporation process employed in this study is that the surface is not homogeneously covered during the initial deposition, in contrast to conventional thin film deposition under high vacuum, where the deposited film homogeneously covers the substrate from the beginning. As indicated in One of the significant features of plasmonic nanostructures with high SERS activity is the existence of nano-gaps, which are formed among the Au nanoparticle aggregates in this study. This means that the SERS activity should be reduced if we suppress the formation of the nanogaps or remove the existing nano-gaps, as displayed in In addition to the annealing effect of the plasmonic nanostructures, the resistance of nanoporosity to liquid environment was also assessed. Agglomeration occurred in solvent such as water and ethanol causing the reduction of the nanoporosity. The degree of agglomeration was much higher in water than in ethanol. However, in hexane, the shape of nanoparticle aggregates and nanoporous morphology was still maintained. While the effects of SERS performance induced by liquid solvents were not investigated, both agglomerated and non-agglomerated nanostructures are expected to have comparable SERS sensitivity.When physical vapor deposition, such as evaporation, is considered for the fabrication of thin films, it is usually understood that atoms move from the deposition source to the substrates without collision under high vacuum. By simply increasing the pressure, a highly porous structure can be synthesized through the homogeneous nucleation and growth of evaporated atoms. The obtained porous structures consist of nanoparticle aggregates with nano-gaps among the particles. These plasmonic nanostructures enhanced the Raman signal intensity as they were used for SERS, outperforming commercial SERS substrates. A simulation using the finite-difference time-domain (FDTD) method, which is under investigation, will elucidate the change of photophysical surface plasmonic resonance energy in terms of various morphologies and light absorption. With further studies on the optimization of the thickness of the deposited porous layer, the size of individual nanoparticles, porosity owing to pressure control, and high-pressure thermal-evaporation-based nanostructures explored in this study could provide a cost-effective alternative to chemical sensors.


Cartilage damage has a certain ability to spontaneously repair, but the repaired tissue often shows the phenomenon of cartilage terminal differentiation, which causes irreversible damage to its structure and function and seriously affects the quality of life and work of patients. It is of great significance to study the problems encountered in the process of cartilage damage repair. This article mainly studied stem cells to promote the regeneration of knee joint degenerative bone articular cartilage. First, the animal articular cartilage defect is modeled, 10 ml of animal venous blood is drawn, 0.5 ml of PRP is collected by centrifugation, mixed with cartilage fragments, and transplanted into the defect area into a gel. In the BMSCs group, 1 ml of BMSCs with a cell concentration of 107 cells/ml was injected intra-articularly. The histological chromosomes were observed after 6 weeks and 12 weeks, and the effect of cartilage tissue repair was analyzed and evaluated, and the related data were statistically analyzed. We evaluated the spontaneous repair ability of partial cartilage damage, full-thickness cartilage damage, and osteochondral damage. Furthermore, for partial cartilage damage repair, by using the cartilage damage in vitro model and biomaterials to simulate the in vivo microenvironment, the adhesion and cell morphology on the surface of partial- and full-thickness cartilage damage were evaluated, and the experiments were further used to evaluate the exogenous and internal induced migration effect of source on cultured cells in vitro. In the cell concentration study, the cartilage repair effect increased with the increase in concentration within a certain range, and the tissue repair ability remained stable when the concentration exceeded 107 cells/ml. Using ECM-oriented scaffolds to compound autologous BMSCs, tissue-engineered cartilage was successfully constructed, which had the histological and biochemical characteristics of normal cartilage tissue, and better repaired the damaged articular cartilage of large animals. Joint degeneration is usually caused by trauma or other pathological causes of cartilage and osteochondral damage, which ultimately leads to the complete destruction of the entire joint structure, resulting in total loss of joint function. Current research shows that although certain biological agents can delay the deterioration of the disease and reduce the pain of patients to varying degrees, these drugs cannot effectively promote tissue regeneration. Due to the extremely limited ability of articular cartilage regeneration, arthroplasty has become an inevitable surgical method to restore joint function in the late stage. This review describes the clinical repair techniques widely used to repair defects in articular cartilage and discusses the long-term and short-term clinical effects of these techniques. At the same time, in the next 10 years, the combination of therapeutic methods and the development of regenerative biologics is expected to cure joint cartilage damage and restore joint function. These products include cell-based and cell-free materials, such as autologous and allogeneic cells, and a variety of versatile cell-based technologies. The key to these technologies is to use tissue engineering methods to transform biotechnology into clinical products, thus playing an important role in these aspects.Articular cartilage injuries are very widespread, such as trauma, genetics, and degenerative changes. In the process of postinjury repair, what factors are at work? A large number of experimental studies have proved that there are indeed some proteins in the process of articular cartilage repair that play an important role in the regulation of cartilage metabolism , but theα-stimulated OA chondrocyte monolayer culture with BMMSC-EVs; 48h later, used qRT-PCR to detect proinflammatory gene-expressing chondrocytes; and 4 weeks later, 1, 9-dimethylmethylene blue (DMMB) analyzed its glycosaminoglycan content. In addition, paraffin sections of the regenerated tissue were stained for proteoglycan (saffron-O) and type II collagen (immunostaining). His method proved that BMMSC-EVs are an important medium for cartilage repair, but the detailed principle is still not proven [Osteoarthritis (OA) is a rheumatic disease that causes chronic pain and disability. There is no effective treatment. Vonk LA studied the role of extracellular vesicles (EVs) secreted by human bone marrow mesenchymal stem cells (BMMSCs) in the repair of human OA cartilage. He tested and observed the effect of BMMSC-EVs on OA cartilage inflammation; treated TNF-t proven .Cartilage tissue engineering is an optimized combination of a new type of scaffold, cell source, and growth factors and has become a promising strategy for cartilage regeneration and repair. Fu N. studied the role of polycaprolactone-polyethylene glycol-polycaprolactone (PCL-PEG-PCL) PCEC scaffold in cartilage repair. He first prepared PCEC thin films and tested their properties using scanning electron microscopy and atomic force microscopy. After cell inoculation, the morphology of cells on the PCEC membrane (SCC rat adipose-derived stem cells and muscs green fluorescent mouse adipose stem cells) was observed by scanning electron microscopy and fluorescence microscopy. The Ccts-8 method was used to detect cell viability on PCEC membranes. In addition, complete cartilage defects were created in rats, and the PCEC film was embedded to evaluate its healing effect for 8 weeks. His method is one-sided and does not objectively explain the results of the experiment . Bone maThis study tested the effects of BMSC-Exos and articular chondrocyte-derived exosomes (AC-Exos) on cartilage regeneration through a series of experiments. PIC hydrogel is used as a biological carrier, loaded with BMSC-Exos and AC-Exos, and the repair of sheep's articular cartilage defects is used as an animal model. The in vivo experiment was evaluated by gross ICRS, cartilage repair score, histological staining, and immunohistochemical staining. The effect of different exosomes in repairing cartilage damage and the degree of fibrosis of new tissues were observed; in vitro experiments were conducted to coculture BMSC-Exos and AC-Exos with chondrocytes and observed using a light microscope, western blot, flow cytometry, and immunofluorescence technology to detect changes in the phenotype of chondrocytes.There are four main aspects that affect the quality of life of the elderly, including physical health, mental health, and external economic and social activities. The most important thing for the elderly is physical health. Since birth, old age, sickness, and death are the normal natural aging processes of people; when there is a disease, it will directly affect the health of the elderly. Because it is not healthy enough, their natural aging process will naturally accelerate, which is directly reflected in the gradual degradation of various body functions, so that the physical and mental health of the elderly suffer a double blow. Degenerative musculoskeletal disease and other diseases have a high incidence in the elderly .The structure of the knee joint is more complex, with the upper femur and the lower tibial plateau, as well as the overlying ligament muscle tissue, so the degenerative changes of the muscles around the knee joint will cause corresponding changes in the knee joint structure. Studies have shown that people lose 1% to 3% of their muscle strength every day without exercise . In eldeDegenerative knee pain has become a common phenomenon. Degenerative knee pain is a major factor affecting the quality of life of the elderly. It often manifests as pain when going up and down stairs, walking, unstable and bumpy, and poor flexion and extension. In severe cases, loss of joint function may occur, leading to loss of labor and living ability , 10. TheAccording to the composition of the matrix, cartilage can be divided into elastic cartilage, fibrocartilage, and hyaline cartilage. Articular cartilage is hyaline cartilage tissue that grows on the surface of the joint. Hyaline articular cartilage is an avascular and nerveless tissue composed of chondrocytes, water, collagen fibers, and proteoglycans .The emergence of tissue engineering technology has brought another possibility for the repair and reconstruction of organs and tissues . Cartilaβ, PDGF-BB, FGF, BMP, and IGF, have been used in cartilage tissue engineering [Cartilage tissue engineering includes cartilage seed cells, scaffolds, and sensor signals. Cells from various sources have been used as seed cells for cartilage tissue engineering, such as chondrocytes, fibroblasts, and stem cells. Regulatory signals such as growth factors have the effect of accelerating and promoting cartilage repair. Growth factors can be added to the culture medium to promote cell growth and differentiation when cells are cultured in vitro, and growth factors can also be loaded into the scaffold to promote cell differentiation and organization of regeneration. Many growth factors, such as TGF-ineering .Although the method of tissue engineering has been successful in the regeneration of many soft tissues, there are still many unsolved problems in cartilage tissue engineering. On the one hand, the structure and properties of the cartilage itself make it difficult to repair itself after injury. On the other hand, how to find a more advanced, more convenient, and more controllable three-dimensional scaffold preparation technology is particularly important. The prepared tissue-engineered cartilage scaffold can simulate the internal structure and physiological composition of natural cartilage, to give cells and tissues the best growth microenvironment. There are still challenges to achieve the ideal functional replacement.β1 that maintain the shape of chondrocytes in a three-dimensional scaffold. Another notable feature is their ability to selectively migrate to diseased tissues and organs. They are found to secrete cytokines that inhibit cell proliferation in cartilage defects. These cytokines are thought to play an essential role in allograft and xenotransplantation. In the context of cartilage repair, MSCs can activate specific tissue repair systems that can be used to nourish cartilage and promote its growth and repair by directly releasing synthetic, proliferative, and regenerative factors into cartilage damage. Through the release of chemokines to create a regenerative environment, bone marrow mesenchymal stem cells may also drive endogenous stem cells into the cartilage-damaged area to repair damaged cartilage tissue [Among the various types of stem cells in the human body, stem cells extracted from the bone marrow have many unique advantages in clinical applications. Due to their relatively easy separation and strong reproductive capacity, it is convenient for us to obtain them from donors and store them in readymade cell banks. In addition, clinical experiments have confirmed that a large number of clinical experiments conducted in vitro and in vivo have found that human bone marrow mesenchymal stem cells can differentiate into cartilage tissue and bone tissue and play an important role in the regeneration of osteochondral defects and cartilage defects . Specifie tissue .At present, most studies on cartilage regeneration through stem cells are bone marrow-derived mesenchymal stem cells. Although the extraction process of the bone marrow is very painful, it is an effective method to obtain stem cells. However, stem cells can also be obtained from other sources, including adipose tissue, muscle, cortical cancellous bone, synovium, periosteum, and umbilical cord. Human adipose stem cells are the easiest to obtain. In vitro studies have shown that these stem cells have proliferation and differentiation capabilities similar to bone marrow mesenchymal stem cells. Adipose-derived stem cells are considered an ideal source of stem cells due to their relatively strong in vitro isolation ability, anti-inflammatory properties, and no immunogenicity. Compared with stem cells from other sources, their proliferation and differentiation abilities are unlikely to be affected by individuals. The effect of age: by injecting adipose stem cells into the knee joints of elderly patients suffering from knee arthritis, combined with arthroscopic minimally invasive surgery for joint cavity lavage, the clinical feasibility and safety of human adipose stem cells in the treatment of degenerative cartilage diseases have been studied. The result of the treatment is to improve body function and promote articular cartilage repair, thereby alleviating the pain of the patient's knee joint . HoweverThe oriented scaffold facilitates the adhesion and growth of seed cells in the scaffold, facilitates the uniform distribution of seed cells inside the scaffold, and helps the seed cells arrange in the scaffold into the arrangement of cells in natural articular cartilage. This determines the structure and functional characteristics of the new organization. This is of great significance for the construction of functional tissue-engineered cartilage. In previous studies, the oriented scaffold combined with autologous bone marrow stem cells can better repair rabbit knee joints after cartilage orientation, but there is no report on its use in repairing articular cartilage defects in large animals.Bone marrow stromal stem cells have multidirectional differentiation potential and convenient sources. Cells derived from autologous sources have no effect on the body's donor area. They can proliferate rapidly in vitro and will not lose their stem cell characteristics after multiple passages. They are currently available in cartilage tissue engineering, one of the sources of seed cells of concern. However, the previous research work was based on the chondrogenic induction of small animal bone marrow stem cells, and the long-term effect of the induction has not been evaluated for safety. Therefore, this study selected autologous bone marrow stromal stem cells as seed cells and repaired sheep's articular cartilage defects without any inducing factors after the composite orientation scaffold.The obtained cartilage tissue was send to the clinical cell culture room for cultivation. In strict accordance with the requirements and standards of the China Pharmaceutical and Biological Products Inspection Institute, the autologous chondrocytes are separated, cultured, expanded, and quality monitored. The cultivation period is 14–21 days. The isolated primary cells were expanded to 107 at low density. Seed cells were in vitro. Composite three-dimensional scaffold and the operation follows the standardized operation process of tissue-engineered cartilage transplantation.They were cultured in vitro for a total of 2-3 weeks, a second small incision transplantation operation was performed, and the constructed tissue-engineered cartilage tissue was implanted back into the defect site. The operation followed the standard operating procedure of tissue-engineered cartilage transplantation. On the day of surgery, the tissue-engineered cartilage tissue is transferred to the hospital, the cell biochemical function is tested at the same time, and the culture solution is tested for bacteria and mycoplasma to ensure the quality of the implanted tissue and is free from contamination. Surgical method: Make a small incision to cut the joints, use a sharp blade to cut vertically around the damaged cartilage to the subchondral bone, and use a circular curette to debride the damaged subchondral bone tissue. Trim the tissue to be transplanted into a defect shape, and fix the tissue-engineered cartilage tissue on the edge of the defect with a 6–0 threaded needle absorbable suture . Seal the suture area with fibrin glue. Finally, check the graft fixation, passively extend and flex the joint several times, and suture the wound after confirming that there is no displacement.The mechanical test is used to evaluate the compression performance of the repaired tissue. First, the sample is fixed to the base of the mechanical tester , and the position is adjusted so that the center of the defect is directly below the compression mold. The compression test is performed with a force of 10N. The speed is set at 0.06 mm/min. When the cartilage layer is compressed to 20% of the thickness of the cartilage layer, the compression is stopped and the compression is allowed to return to the original position. The slope of the stress-strain curve at this time is the measured compressive modulus (MPa). Before the mechanical test, the frozen samples are placed in PBS at room temperature for 3-4 hours to fully equilibrate.Observe the general shape, color, texture, and volume changes of the cell-material complex after one week. Observe the survival and distribution of cells in the cell-material complex after one week of culture. Take the cell scaffold complex that has been cultured for one week, rinse with PBS, and immediately freeze the sections. The sections will be air-dried and fixed with 95% alcohol for AO-PI staining and Hoechst 33258 fluorescent staining; observe the bone marrow stem cells on the scaffold under an inverted microscope.We stained the envelopes of BMSC-Exos and AC-Exos with green and red fluorescence with membrane fluorescent dyes and cocultured them with chondrocytes. Through fluorescence microscopy, we found that a large number of green and red fluorescences were accumulated in the cytoplasm of chondrocytes. It is suggested that both exosomes can be taken up by chondrocytes in large quantities, and the two exosomes can enter chondrocytes to play a role .When chondrocytes are cultured in vitro, they will gradually dedifferentiate as the number of culture passages increases, losing the unique phenotype of chondrocytes. The tissue obtained by expansion of such dedifferentiated chondrocytes is usually fibrocartilage . The norAs shown in The results of immunofluorescence staining showed that the expression of Aggrecan (red fluorescence) of chondrocytes treated with BMSC-Exos was significantly reduced from the 4th generation, while for chondrocytes treated with AC-Exos, the expression of Aggrecan always remained at a higher level.μg/ml, and the average cellular iron content was 2.77 ± 0.93 mmol/L. When SPIO continued to increase, the intracellular iron showed a downward trend [μg/ml and 12 μg/ml) will reduce the uptake of SPIO by cells.As shown in rd trend . Throughμg/ml or more and the cells are cocultured for 24 hours, the positive rate is found by the microscope after staining with Prussian blue. Up to 99%, it proves that the new SPIO has been successfully transferred into BMSCs with extremely high labeling efficiency [As shown in ficiency . ExperimAs shown in As shown in We further studied the regulation effect of EHG hydrogel on the cells surrounding the defect through in vivo experiments. The results of DAPI staining showed that the number of cells in the defect in the EHG group was significantly higher than that in the HG group. The results of HE staining showed that chondrocytes, BMSCs, and inflammatory cells were seen in the defect. This result suggests that EHG hydrogels can efficiently recruit chondrocytes and BSMCs around the defect into the defect site.Cartilage tissue cannot heal by itself after injury, and autologous chondrocyte transplantation is limited by the lack of cell source and quantity. From the perspective of tissue-specific stem cells, this study confirmed that cartilage tissue has stem cell characteristics and high cell proliferation efficiency regardless of physiological and pathological conditions. The appearance of such cells may be due to changes in the environment, and related surface markers are dynamically changing.Experiments in this article confirm that hiPS-MSC-Exos can effectively repair cartilage damage and promote hyaline cartilage regeneration. The results of in vitro experiments confirmed that hiPS-MSC-Exos can significantly enhance the proliferation and migration of chondrocytes and BMSCs. This result suggests that hiPS-MSC-Exos has the function of significantly promoting cartilage cell regeneration and repairing cartilage damage, showing a broad transformation application prospect.Part of the cartilage is damaged, and its matrix microenvironment is not easy for cells to adhere to and lacks small biological molecules that can effectively induce cell migration. Therefore, we use biomembrane materials combined with matrix cell-derived factors to construct bioactive materials to promote and adhere to migration and then open cartilage repair spontaneously; in osteochondral damage, there are a large number of bone marrow-derived seed cells in the matrix microenvironment to participate in the repair, but in the environment of osteochondral damage, the bone system is more inclined to differentiate, resulting in abnormal cartilage terminal differentiation. Therefore, we used collagen fragrant silk double-layer scaffolds combined with parathyroid hormone-related proteins to inhibit the terminal differentiation of cartilage, promote the process of cartilage formation, and improve the quality of spontaneous cartilage repair.