The emergence of introns was a significant evolutionary leap that is a major distinguishing feature between prokaryotic and eukaryotic genomes. While historically introns were regarded merely as the sequences that are removed to produce spliced transcripts encoding functional products, increasingly data suggests that introns play important roles in the regulation of gene expression. Here, we use an intron-centric lens to review the role of introns in eukaryotic gene expression. First, we focus on intron architecture and how it may influence mechanisms of splicing. Second, we focus on the implications of spliceosomal snRNAs and their variants on intron splicing. Finally, we discuss how the presence of introns and the need to splice them influences transcription regulation. Despite the abundance of introns in the eukaryotic genome and their emerging role regulating gene expression, a lot remains unexplored. Therefore, here we refer to introns as the “dark matter” of the eukaryotic genome and discuss some of the outstanding questions in the field. Historically, introns were considered the non-coding, non-functional sequence elements which disrupt those that are protein-coding, called exons . While tIntrons date back to the last eukaryotic common ancestor, after invasion into the early eukaryotic genome . While a. Initially characterized in the maize genome, transposable elements are repetitive sequences found across eukaryotes and are critically known for their ability to relocate in the genome and alter gene expression  are transcribed by RNA polymerase II, while U6 and U6atac expression are largely dependent on RNA polymerase III . InitiatCLASP1 . TherefoWithin the genome, spliceosomal snRNAs often exist both as gene copies and gene families, whereby divergent genes can encode for variant snRNAs with nucleotide polymorphisms . There aDrosophila, and Lytechinus variegatus (Perhaps counterintuitively, U5 snRNA has the smallest gene family, yet it is the only shared snRNA between the major and minor spliceosomes. Investigations by riegatus . The expriegatus .trans-spliced introns.Functional sequence variants of the snRNAs have the potential to contact cryptic or degenerating splice sites, make novel protein interactions, and adopt secondary structures that alter spliceosome conformation. It is thus possible, given our evolving understanding of consensus sequences, that these variant snRNAs do confer complementarity to specific intron splice sites. Accordingly, from an intron-centric perspective, we must allow for the possibility that seemingly unproductive snRNAs are leveraged to splice a specific subset of introns. A role for non-consensus intron classes was voiced by RNU12 which is causal to early onset cerebellar ataxia (It remains to be established if variant snRNAs are evolutionarily selected for use in differential splicing or if they arise stochastically. Though, one could imagine that selective use of a variant splicing component would provide an opportunity to splice novel or divergent splice site sequences. It is known that mutations in the snRNAs can have pathogenic effects, as demonstrated by r ataxia . Additior ataxia . In anotr ataxia . RegardlIntrons have served a valuable evolutionary role for eukaryotes in that they are more prone to genetic drift compared to exons. Introns appear to be under weaker selection than exons in somatic cells, which may be due to a mismatch repair system employed for exons that is notably lacking for introns . Using aSaccharomyces cerevisiae (S. cerevisiae compared to other species, such as vertebrates and land plants, making the yeast genome a strong model for intron depletion studies (. Indeed, S. cerevisiae could survive without introns, however, intron-depleted strains fared variably when subjected to drug-induced and carbon source stresses. However, transcription machinery was found to be capable of responding to expression deficits following intron-depletion by using alternative promoter selections, highlighting the role introns play in expanding the eukaryotic transcriptome (S. cerevisiae cells under antibiotic selection and subjected to a lab-evolution paradigm. Growth and transcriptomic analyses of derived cell generations revealed independent, adaptive mutations occurring both cis- and trans-to improve splicing efficiency and thus antibiotic resistance and cell survivability. The cis-mutations were proposed to increase accessibility of splice site sequences, while trans-mutations might increase the cellular abundance of splicing machinery. Importantly, cis-fitness-inducing mutations could alleviate selection-independent splicing inefficiencies, however, mutations in trans-were particularly advantageous during periods of active selection (S. cerevisiae, one can imagine that similar mechanisms may be employed for evolutionary adaptation. For example, in ecotypic Cichlid fish, alternative splicing is a dominant mechanism for rapid changes in gene expression. Specifically, alternative splicing underpins the diversification of jaw morphology as it relates to the food they have evolved to consume in different ecological niches (While introns are seemingly advantageous, prokaryotes show that the absence of introns is not prohibitive to life. This begs the question, to what extent do eukaryotic cells really require introns? In one study, revisiae . Introns studies . Indeed,criptome . Should criptome . To testelection . Though l niches .In both mammals and plants, the presence of introns is known to enhance gene expression in a phenomenon sometimes referred to as intron-mediated enhancement . The recThe mechanism by which 5’ introns regulate transcription involves, at least in part, control of the open chromatin signatures H3K4me3 and H3K9ac, which facilitate recruitment of RNA polymerase II and general transcription factors to promoters. These marks are deposited at the first exon-intron boundary of genes, explaining how the distance between transcription start site and the first intron can influence the expression level of a gene . While aOne caveat to the spliceostatin A experiment is that it inhibits the entire splicing machinery, without revealing the specific interactions between the spliceosome and intron consensus sequences that enhance transcription. In fact, it is not the entire spliceosome that needs to be activated for transcription enhancement, as the formation of stable interactions between U1 snRNA and the promoter-proximal 5′SS can enhance transcription . RecruitBeside the role of U1 in transcription initiation, U1 snRNA is also independently involved in preventing pre-mature transcription termination, which can occur if RNA polymerase II encounters a polyadenylation site within an intron. Surmounting 3′ end sequencing data has revealed that introns often contain cryptic or pre-mature polyadenylation sites that result in the destabilization of RNA polymerase II, thereby producing truncated transcripts incapable of encoding a protein . Remarkain situ hybridization analyses, as well as RNA and protein blotting of subcellular compartment extracts (Genes, chromatin, and RNA polymerase II have a subnuclear organization around topologically-associated domains to phase-separate euchromatic regions of active transcription . Alongsiextracts . While textracts . Punctatextracts . For exaextracts .In all, here through an intron-centric lens, we focus our attention on the myriad of regulatory and functional consequences that have emerged by the presence of introns in the genome. Thus, we hope that future studies will begin to shed light on this “dark matter” of the eukaryotic genome to uncover the secrets buried within. Importantly, the advent of next-generation sequencing and computational analysis will invariably play a critical role in uncovering some of these mysteries. Throughout this article, we have described several of these methods, and here we point readers to other reviews .


N = 152), aged 9;0–10;0. To measure the children’s vocabulary the PPVT was used in Dutch (Schlichting, Second language proficiency may be related to first language acquisition (Ganschow & Sparks,  Accounting for individual differences in language learning has been crucial to the field of second language acquisition (SLA). It is well established that there is a continuum of language learning, which spans from very poor second language learners to nativelike foreign language users  skills and foreign language (FL) learning  were excluded, as were the data from children who had a mother tongue other than Dutch (N = 123). The reason for these exclusions was that we carefully wanted to examine the relation between Dutch and English, without interference from other languages or language impairments. Other than obtaining on average lower scores on the Dutch tasks, these excluded children did not behave very differently as a group from the participants in the present sample. Finally, some participants did not complete all of the relevant tasks and those data were excluded, too (N = 7). Hence, the total number of participants was 152. There were 76 male and 76 female participants. The mean age of the participants was 9 years and 10 months. The participants were tested half-way through the school year.Two hundred and ninety-eight grade 4 pupils across seven primary schools in the Randstad, the conurbation in the western part of the Netherlands, took part in the first round of data collection. Parental permission (active consent) was obtained for each participant. The study was approved by the Ethical Review Board of the university. The schools had different pupil populations regarding social-economic status based on their postal codes and regarding the number of children with Dutch as an additional language based on the answers the participants gave in the language background questionnaires. Two schools were situated in an urban area, four in a suburban area and one in a rural area. According to national Dutch normative data, the schools were situated in neighbourhoods from low to middle social-economic status SCP, . Hence, NRAVEN = 60). Each item consists of a geometric pattern with one piece missing. Participants have to discover the principles, relationships and rules between the patterns and decide which of six given pieces is the missing one. A test-retest reliability of 0.88 was reported for the test , regardless of how many mistakes they made in a set.The measure of L1 vocabulary was the Peabody Picture Vocabulary Test, Third edition . This test was administered to assess the participants’ ability to formulate grammatically acceptable and semantically meaningful sentences by manipulating and transforming given words and word groups. Almost all of the words in the sentence assembly task were high frequent, basic words. L1 sentence production was examined by using the task Sentence Assembly from the Clinical Evaluation of Language Fundamentals [hij] [weet] [niet] [of] [hij het][verstuurd heeft].[he] [knows] [not] [if] [he it] [sent has]‘He does not know whether he sent it.’(3)[weet] [hij] [niet] [of] [hij het] [verstuurd heeft].[knows] [he] [not] [if] [he it] [sent has]‘Does he not know whether he sent it?’This task was used to assess L1 grammar knowledge in previous studies , regardless of how many mistakes they made in a set, because the test was administered in class.The measure of FL vocabulary was the Peabody Picture Vocabulary Test, Fourth edition . The test was administered to assess the participants’ ability to interpret spoken sentences and to select the pictures that illustrate referential meaning of the sentences. This test was used to assess L1 grammatical knowledge in previous studies . The present administration differed slightly from standard administration. Participants were presented with a coloured picture and a sound file containing the target sentence. The sound file was recorded by a female native speaker of British English. Participants were asked to complete the target sentence in English. All of the instructions were in Dutch. Finally, participants received one point for each correct answer, leading to a score out of 12.Word Structure from the Clinical Evaluation of Language Fundamentals , so that the participants’ answers were recorded and stored automatically and digitally. Each class session took about 45–60 min to complete. The language background and the English (FL) morpho-syntax tests were administered individually. Individual sessions took about 30 min to complete.All of the tests were part of a larger test battery in a three-year longitudinal study into the predictors of English language learning by Dutch primary school pupils. Testing began in February for four schools, in March for two schools and in April for one school and lasted about three months, depending on the availability of the school. The test battery consisted of class sessions, in which participants worked by themselves in their classroom, and individual sessions, in which a test administrator tested a participant individually in a separate room. In the larger test battery, there were five class sessions and six individual sessions. Participants carried out the tests by themselves on a tablet in the class sessions; they wore headphones and a short video served as an instruction for each test. Three of the class sessions contained Dutch (L1) language tests and the other two included English (FL) language tests. Each participant had a tablet  to work on. The Dutch (L1) and English (FL) vocabulary tests, the Dutch (L1) sentence assembly and the English (FL) oral comprehension test were part of the class sessions. All the class tests were converted to an online survey (The mean scores of the 152 participants on the Raven and the Dutch (L1) and English (FL) measures are reported in Table r correlation analysis. Table r correlation values for all the L1 and FL measures.First we wanted to know whether L1 vocabulary and L1 grammar were related, as our first research question asked: are L1 vocabulary and grammar related in children who have outgrown the early stage of grammatical development? To determine whether this relationship held, we carried out a Pearson r = .33, p < .001), meaning that participants who had a larger vocabulary in Dutch, also performed better on L1 sentence assembly, and vice versa.Table r = .64, p < .001) and between FL vocabulary and FL morpho-syntax , indicating that participants who had a larger vocabulary in English, also performed better on the English grammar tasks, and vice versa.We also wanted to know whether FL vocabulary and FL grammar were related. Our second research question asked: Are FL vocabulary and grammar related in young, beginning learners in an educational setting? Table p values were obtained with the lmerTest package . A summary of the results of both models can be seen in Table p = .029, z = 2.185, with a 95% confidence interval from 1.002 to 1.032). FL vocabulary increases the odd-ratio of scoring more correctly in the FL oral comprehension test by 1.062. This difference is significant . Next, we look at FL morpho-syntax, as the third research question asked whether L1 vocabulary, L1 grammatical knowledge, logical reasoning and FL vocabulary was associated with FL grammatical knowledge. The results of the model with FL morpho-syntax as the dependent variable show that L1 grammatical knowledge, L1 vocabulary and FL vocabulary increase the odd-ratio of having more correct scores on the FL morpho-syntax task. Specifically, L1 grammatical knowledge increases the odd-ratio of scoring more correctly on the FL morpho-syntax task by 1.017. This effect is significant . L1 vocabulary increases the odd-ratio of obtaining more correct scores on the FL morpho-syntax task by 1.04. This effect is significant . Finally, the strongest predictor of the FL morpho-syntax task is FL vocabulary, which increases the odd-ratio of obtaining more correct scores in the FL morpho-syntax task by 1.12. This effect is significant .The third research question asked whether L1 vocabulary, L1 grammatical knowledge, logical reasoning and FL vocabulary predicted FL grammatical knowledge. First we look at FL oral comprehension. The results of the model with FL oral comprehension as dependent variable show that only L1 vocabulary and FL vocabulary are significant predictors of the score participants obtained in the FL oral comprehension test. Specifically, L1 vocabulary scores increase the odd-ratio of scoring more correctly in the FL oral comprehension task by 1.017. This effect was statistically significant  appeared in the vocabulary task and in the FL oral comprehension task and/or in the FL morpho-syntactic task. Hence, the FL tasks differed in the words they employed. Moreover, the grammar tasks were designed to assess grammatical knowledge: the outcomes are a representation of how much syntactic knowledge of English these Dutch nine-year-old pupils have at their disposal. Although vocabulary was involved in the grammar tasks, they measured grammatical knowledge. Based on our findings, again the conclusion can be drawn that language experience is key. The more FL learners have been exposed to words, the more likely it is that they also have more experience with diverse grammatical structures (see for a similar account for young children Hoff et al., A positive relationship was also expected between FL vocabulary and FL grammar. A strong correlation with vocabulary was observed for both the FL oral comprehension task, assessing receptive syntactic knowledge, and the FL morpho-syntactic task, assessing productive morphological knowledge. This finding suggests that the Dutch pupils who knew more words on the English vocabulary task were also significantly more likely to understand English sentences correctly and more likely to complete English sentences correctly than their peers who knew fewer words. This is in line with previous research (Goriot et al., Finally, it was hypothesised that FL grammar would be predicted by L1 vocabulary, L1 grammar and FL vocabulary. For the FL oral comprehension task it was found that both FL vocabulary and L1 vocabulary were significant predictors of the English oral comprehension task. This pattern indicates that the variation in the performance on the English oral comprehension task could, to a certain extent, be predicted by Dutch children’s English and Dutch vocabulary knowledge. A plausible explanation for this finding is that oral comprehension depends not only on grammatical comprehension but also on lexical comprehension. The choice of task may thus explain the absence of transfer of Dutch grammar to English oral comprehension. Kohnert et al. , p. 695 The analyses of the FL morpho-syntax task painted a slightly different picture: both Dutch vocabulary and English vocabulary were again significant predictors, but Dutch grammatical knowledge also contributed to the scores on the English morpho-syntax task. Although it has been found that receptive vocabulary can be transferred from the L1 to the FL, children’s overall L1 vocabulary level does not seem to be predictive of FL vocabulary development (Goodrich et al., What conclusion can be drawn from the present findings in light of transfer? Our study shows that this was task dependent: Dutch grammatical proficiency predicted the FL morpho-syntactic task. Dutch vocabulary, which may well be a reflection of morphological skills, also contributed significantly to the English grammar task that tapped into productive morphological knowledge. L1 skills thus lay the foundation for FL proficiency both directly and indirectly. Further research has to be done to understand an effect of task: both the effect of the task used to measure the grammatical abilities in L1 and the tasks used to measure grammatical learning in the FL. In all, our results underline the link between lexical and grammatical representations, as proposed by the framework of usage based grammar: our grammatical measures were related to vocabulary in both the L1 and the FL, and FL morphological knowledge seems mediated by L1 vocabulary knowledge and L1 grammar skills. This means that vocabulary skills are closely linked to grammar skills in an FL and this is already the case for beginning learners in an educational setting.


The potential applications of multilayer graphene in many fields, such as superconductivity and thermal conductivity, continue to emerge. However, there are still many problems in the growth mechanism of multilayer graphene. In this paper, a simple control strategy for the preparation of interlayer-coupled multilayer graphene on a liquid Cu substrate was developed. By adjusting the flow rate of a carrier gas in the CVD system, the effect for finely controlling the carbon source supply was achieved. Therefore, the carbon could diffuse from the edge of the single-layer graphene to underneath the layer of graphene and then interlayer-coupled multilayer graphene with different shapes were prepared. Through a variety of characterization methods, it was determined that the stacked mode of interlayer-coupled multilayer graphene conformed to AB-stacking structure. The small multilayer graphene domains stacked under single-layer graphene was first found, and the growth process and growth mechanism of interlayer-coupled multilayer graphene with winged and umbrella shapes were studied, respectively. This study reveals the growth mechanism of multilayer graphene grown by using a carbon source through edge diffusion, paving the way for the controllable preparation of multilayer graphene on a liquid Cu surface. Graphene has great application prospects in electronics, optics, and biology ,2,3,4. TIn recent years, with the advancement of graphene fabrication techniques, the technology for preparing large-area single-layer graphene using the chemical vapor deposition (CVD) method has become increasingly mature. Research efforts have extended to the fabrication of bilayer and multilayer graphene . For theIn this study, we developed a simple method and control strategy for preparing interlayer-coupled multilayer graphene based on a liquid Cu substrate. By adjusting the gas flow rate in the system, we achieved precise control over the carbon source supply, resulting in the fabrication of interlayer-coupled multilayer graphene with different morphologies. We conducted the first investigation and characterization of morphology and structure of winged interlayer-coupled multilayer graphene. By using characterization techniques, such as SEM, Raman spectroscopy, AFM, and TEM, we confirmed that this type of multilayer graphene exhibits an AB-stacked structure. We also observed the phenomenon of small multilayer graphene stacking beneath single-layer graphene and studied the growth process and mechanism of interlayer-coupled multilayer graphene with different morphologies. This method demonstrates the significant potential of using a liquid Cu surface CVD for the controlled synthesis of multilayer graphene.4, 99.9% purity), hydrogen , argon , and diluted oxygen (5%). Before the graphene growth process, Ar gas was flushed into the quartz tube to remove any trapped air in the system. The furnace was then heated to 1170 °C over a period of 60 min and annealed for 30 min with a flow rate of 100 standard cubic centimeters per minute (sccm) of H2. During the graphene growth stage, specific flow rates of CH4 and Ar gases were introduced into the system to facilitate graphene growth, while the flow rate of hydrogen was adjusted accordingly. Once the growth step was completed, the CH4 gas was switched off, and the furnace was rapidly cooled down to room temperature.To ensure the cleanliness of the Cu foils  and W foils  prior to graphene growth, a series of cleaning steps were performed. First, the foils were immersed in diluted hydrochloric acid, followed by rinsing with deionized water, ethanol, and acetone using ultrasonication. Subsequently, the Cu/W foil was placed inside a quartz tube positioned in the heating zone of a Lindberg/Blue M TF55035A furnace. The growth of graphene was carried out by using atmospheric pressure chemical vapor deposition (CVD) with a mixture of gases, including methane . As shown in To directly observe the morphology and layer number of the graphene, the graphene sample transferred onto SiOI(1-210) is lower than that of the inner-ring diffraction point I(1-100), indicating single-layer graphene. Continuing to observe the diffraction intensity insets in I(1-210) is higher than that of the inner-ring diffraction point I(1-100), suggesting the presence of bilayer or multilayer graphene. This result is consistent with the schematic diagram of the interlayer-coupled multilayer graphene with a tail (To microscopically characterize the layer number of the interlayer-coupled multilayer graphene, the grown graphene sample was transferred onto a coordinate grid for subsequent TEM characterization. As shown in h a tail b. Furtheh a tail c 24]. F. FI(1-21h a tail f 9,15].,15.I(1-2Upon observation, it was found that this interlayer-coupled multilayer graphene exhibited various morphologies. These included the structure with a tail , the strThe presence of different morphologies in interlayer-coupled graphene suggests the formation of multilayer graphene with varying thicknesses in different regions of the graphene. This variation in morphology could be attributed to the carbon source supply during the growth process. Owing to the limited range of control over the methane flow during graphene growth, attempts were made to indirectly regulate the carbon source supply in the actual growth process by adjusting the hydrogen gas flow rate . We fixeFurther investigation was conducted by controlling the carrier gas flow rate to study the effect of gas flow on the preparation of interlayer-coupled multilayer graphene. As shown in 2/Si substrate for further observation, we found that the domains of umbrella-shaped interlayer-coupled multilayer graphene appear significantly darker in color under optical microscopy compared to single-layer graphene (Subsequently, we characterized the umbrella-shaped interlayer-coupled multilayer graphene. As shown in graphene . Based ographene  confirmeI2D/IG ratio (ID/IG Raman mapping map (Raman mapping tests were conducted within the red dashed box region shown in IG ratio  indicateIG ratio  suggestsping map  of the gping map . At highping map . Subsequping map –f. ThereI2D/IG Raman mapping (I2D/IG value greater than 2. However, the I2D/IG values for the inner regions of interlayer-coupled small multilayer graphene are less than 2, indicating that the graphene sample is multilayer in structure. However, from the ID/IG Raman mapping (Although the tail regions of winged-shaped and umbrella-shaped multilayer graphene appear uniform under an optical microscope and SAED patterns in TEM exhibit the AB-stacking structure for both morphologies, a careful examination of the intermediate states of graphene morphology reveals the formation of various regions with different shades beneath the single-layer graphene a. Furthe mapping c, the ou mapping d, it canI2D/IG Raman mapping more clearly shows the presence of small multilayer graphene domains with nonuniform layer thickness at the edges of the single-layer graphene (ID/IG Raman mapping results (I2D/IG (Further observations reveal that during the growth process of graphene, when there is an excess of carbon species, carbon begins to accumulate at the edges of single-layer graphene . As showgraphene . Althouggraphene , the ove results . As the  (I2D/IG  also indFinally, under suitable growth conditions, the formation of winged interlayer-coupled multilayer graphene structures is achieved. As shown in The growth mechanism of umbrella-like, interlayer-coupled multilayer graphene slightly differs from that of wing-like interlayer-coupled multilayer graphene. As shown in We developed a simple method for synthesizing interlayer-coupled multilayer graphene on a liquid Cu substrate under atmospheric pressure. By controlling the Ar gas flow at high temperatures, the “self-limiting” growth mode of single-layer graphene on the Cu surface was broken, resulting in a novel structure of interlayer-coupled multilayer graphene with wing-like features. The morphology characteristics were characterized using SEM, AFM, and optical microscopy, while the layer number and crystallinity were analyzed using Raman spectroscopy and TEM. Additionally, we discovered intermediate states in the growth process of interlayer-coupled multilayer graphene, laying the foundation for further exploration of the growth mechanism of multilayer graphene. This is an exciting development in the field of graphene synthesis.


Currently, sarcoidosis remains one of the diseases with unknown etiology, which significantly complicates its diagnosis and treatment. Various causes of sarcoidosis have been studied for many years. Both organic and inorganic trigger factors, provoking the development of granulomatous inflammation are considered. However, the most promising and evidence-based hypothesis is the development of sarcoidosis as an autoimmune disease, provoked by various adjuvants in genetic predisposed individuals. This concept fits into the structure of the autoimmune/inflammatory syndrome, induced by adjuvants (ASIA) that was proposed in 2011 by Professor Shoenfeld Y. In this paper, the authors reveal the presence of major and minor ASIA criteria for sarcoidosis, propose a new concept of the course of sarcoidosis within the framework of ASIA, and point out the difficulties in creating a model of the disease and the selection of therapy. It is obvious that the data obtained not only bring us closer to understanding the nature of sarcoidosis, but also potentiate new studies confirming this hypothesis by obtaining a model of the disease. According to the current concept, sarcoidosis occurs as a result of exposure to various exogenous or endogenous antigen factors in subjects with a genetic predisposition to autoimmune disorders, and is associated with the development of non-caseating granulomas in different organs .Sarcoidosis is one of the granulomatous diseases with an acute or chronic course. Inflammation most often involves the lungs and mediastinal lymph nodes in up to 90% of cases, as well as other organs and tissues, with granuloma formation without caseous necrosis. Currently, there are two variants of the sarcoidosis acute course: Lofgren’s and the Heerfordt–Waldenström syndrome .The search for etiologic factors of sarcoidosis has revealed various infectious agents (from bacteria to viruses and fungi), as well as non-organic factors  that could be associated with its development .The probThe main key of the pathogenesis of sarcoidosis is the formation of granulomas in the lungs, mediastinal lymph nodes, skin, and other organs .In sarcoidosis, patients with a genetic predisposition to this disease, in which antigen-presenting cells , come into contact with an unknown foreign antigen results in the dysregulated immune response that manifests in granulomatous inflammation .Angiotensin-converting enzyme 2) hyperproduction occurs , and, in general, thyroid autoimmunity ;Sarcoidosis often occurs in conjunction with other autoimmune diseases. The main ones are:Such cases of comorbidity show the possibility of a common pathogenesis of these diseases and confirm the idea of multi-autoimmune diseases .The central part of a granuloma is composed of macrophages, modified macrophages, epithelioid cells, and giant cells, with CD4+ T-lymphocytes between them ,65. The Thus, in patients with neurosarcoidosis, a high CD8 + T-lymphocytes number was found during the immunohistochemical examination of granulomas in autopsies of the meninges and brainstem . And an In addition to CD8 + T-lymphocytes, an increase in CCR4 + CD4 + cell numbers in the peripheral blood was found, as well as an increase in the concentration of the chemokine ligand for the chemokine receptor CCL17 in the blood serum of sarcoidosis patients and locally in the focus of granuloma formation . ExcessiThere is an assumption about the role of the disturbance of the Th1 and Th17 ratio in the formation of granulomas in sarcoidosis . Most reT regulatory cells are immune suppressors and protect against the development of autoimmune diseases. In sarcoidosis, a paradoxical reaction is observed: in the focus of inflammation (in the BAL), a decrease in the number of cells was shown, while in the peripheral blood, to the contrary, an increase was shown. Huang et al. revealed an almost three-fold decrease in the relative number of T-regs in the blood of patients with initially diagnosed sarcoidosis compared with a group of conventionally healthy donors . FurtherIn the study of humoral immunity, it was shown that in patients with sarcoidosis, the amount of B-cell-activating factor (BAFF) is significantly higher and, accordingly, Ig-producing B-cells in the lung tissues, while in the blood the level of B-cells in sarcoidosis, corresponded to the number in healthy people or was below normal . At the According to a comprehensive analysis of B- and T-lymphocytes ,90, sarcA comparison of T- and B-lymphocyte subpopulations in sarcoidosis and autoimmune diseases is presented in We noted that the lymphocyte count can serve as a prognostic factor. The higher number of CD4 cells and a higher CD4/CD8 ratio was observed in radiologically improved patients and responded better to treatment. The increasing number of Th17 cells can be predictive for progressive sarcoidosis and may help in the selection of patients with an increased risk of lung fibrosis . Low neuAt the moment, no specific autoantibodies have been identified in sarcoidosis; however, in various studies, autoantibodies to a wide range of antigens have been found in patients, which is very characteristic of ASIA syndrome . The ShiDS index (1), the use of which suggests the presence of autoimmune inflammation. With an index value of more than seven units (AUC = 0.889) and a sensitivity of 80.56% and a specificity of 93.33%, it is possible to assume the presence of sarcoidosis.According to a comprehensive analysis of antibodies and subpopulations of B-lymphocytes , patient(B-naive\B-memory)—the index of the humoral immune response activity;(CD38-B-cells + CD5-B-cells)—the total number of regulatory B cells (IL-10 synthesis);[anti-MCV]—the concentration of anti-MCV, characterizing the presence of autoimmune reactions.The use of the formula can make it possible to identify patients with the presence of autoimmune inflammation in the pathogenesis of the development of sarcoidosis, which can serve as a recommendation to treat them with immunosuppressive therapy and can serve as a prognostic or predictive marker in assessing the course of the disease and therapy efficacy.Autoantibodies to vimentin and its modifications have been found in various studies in patients with sarcoidosis ,100,101.According to recent studies, HLA genotypes are one of the main genetic predisposing factors for autoimmune disease development. Associations with the HLA-I class, HLA-II, class and non-HLA genes have been identified, e.g., BTNL2, C4, C6 orf10, HSPA1L, LTA, NOTCH4, TAP2, TNF, and VEGF .Löfgren syndrome; HLA-DRB1*01/03, HLA-DQB1*02.01The chronic form of HLA-DQB1*06.02, HLA-DRB1*07/14/15The protective effect of HLA-DRB1*01/04Our greatest interest was in the HLA-DRB1 genotypes, which are associated with the development of many autoimmune diseases. According to the analysis of the distribution of HLA-DRB1, -DQ1 genotypes in different populations  the mainMore detailed data on the distribution of genotypes are presented in It should be noted that most cases of newly diagnosed sarcoidosis do not require treatment, and only dynamic observation is recommended because in half of the patients the disease undergoes spontaneous remission within several years . This asHowever, in some patients, the disease may be accompanied by a progressive course with the formation of insufficiency of the affected organ, which requires therapy. At the same time, despite the treatment, in a significant number of patients the sarcoidosis will turn into a chronic and progressive form, and 6–7% will be fatal . One of One of the features of sarcoidosis that links it to autoimmune diseases is the response to the therapy commonly used to treat these diseases ,125. No At the moment, the first line of therapy includes glucocorticosteroids (prednisolone and methylprednisolone) . AlternaIn case of the ineffectiveness of the second line of therapy, the next step is to prescribe biological therapy drugs . TargeteIn the case of contraindications or ineffectiveness, it is possible to use off-label hydroxychloroquine, as well as mycophenolate, cyclophosphamide, or rituximab .M. Tuberculosis, P. Acne, carbon nanoparticles) in mouse models, only pulmonary granulomas without fibrosis were obtained. Genetically modified mice developed multiple sites of granulomatosis , which may indicate the peculiarities of the immune response in healthy mice, allowing for the more efficient elimination of pathogens [Despite the long-term studies on the pathogenesis of sarcoidosis, there are still no reliable models of the disease , which wathogens . It was In a recent publications, it was shown in a mice model that in vimentin-immunization mice granulomas were found in the lung following intervenous challenge with vimentin-coated beads . SarcoidThe unambiguous conclusion of all existing reviews and studies devoted to the creation of a model of sarcoidosis is that in the present conditions it is impossible to achieve this goal, which determines the empirical approach to treatment.connection with the HLA genotype;the impact of various trigger factors;improvement of the condition with the elimination of the trigger factor;immunological disorders in the form of cytotoxicity of lymphocytes upon stimulation with autoantigens, the presence of autoantibodies, an imbalance of subpopulations of T- and B-lymphocytes typical for many autoimmune diseases;clinical manifestations characteristic of autoimmune pathology;a histological picture of the disease;an association with other autoimmune diseases;the efficacy of immunosuppressive therapy.According to the analysis, it can be assumed that there are several autoimmune features in the concept of ASIA syndrome in sarcoidosis. The characteristic features confirming this fact include :connectiHowever, despite certain associations between sarcoidosis and ASIA, it is impossible to unambiguously consider all variants of the course of sarcoidosis within the framework of this syndrome. Perhaps we should talk about different diseases or different “stages” of the course of sarcoidosis, depending on the continuing influence of the trigger factor. The majority of patients with sarcoidosis are prone to self-healing that can be associated not only with genetic and immunological characteristics.With regard to the relationship between ASIA and sarcoidosis, it is important to identify a cohort of patients in whom self-healing does not occur, and the course of the disease becomes progressive with the development of fibrosis, organ failure, and the generalization of the process. We can assume that in this situation the pathoantigen is not eliminated, and a full-fledged autoimmune disease develops, requiring the appointment of immunosuppressive therapy, which fits into the concept and criteria of ASIA .The analysis of the connection between ASIA and sarcoidosis suggests the need to continue research with studying the prognostic and predictive factors that determine the course of the disease, in addition to treatment strategies. Questions about the etiological factors, the creation of a model of sarcoidosis, and the justified prescription of immunosuppressives, including biological therapy, remain unanswered. And the relationship of sarcoidosis and ASIA, on the contrary, raises new ones.


Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem  classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3′ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen  A)  is a growing health and economic threat and has been estimated to be directly responsible for 1.27 million deaths worldwide in 2019 . ExtensiFig. 1A) , 5.FigurA)  , for example, cefoxitin and cefmetazole, are referred to as 7α-methoxy cephalosporins  containing an oxyimino group are referred to as third-generation- or extended-spectrum oxyimino-cephalosporins and have an extended spectrum of activity compared to earlier cephalosporins  . CephemsA) . They arFig. 1A) , 9. Cephosporins , second-osporins . Those tosporins . Clinicaosporins . Only onosporins , 15. Theosporins . The carins 2 in , 17.c. 3.6 Å) zinc ions, termed Zn1 and Zn2, bridged by the hydroxide nucleophile, thought, respectively, to activate the β-lactam carbonyl for nucleophilic addition and stabilize accumulating negative charge on the β-lactam amide N.The efficacy of all β-lactams is increasingly being diminished due to the emergence and reemergence of β-lactamases that hydrolyze the β-lactam ring to render them inactive. β-Lactamase enzymes are grouped into four main classes : A, C, aB) the β-lactam N is then protonated, resulting in the formation of an inactive penicilloic acid. Cephem hydrolysis can result in different products (C). This mechanism of cephem product generation, involving loss of the 3′ leaving group, has been exploited in the development of fluorogenic reporter substrates for SBLs and MBLs (2-enamine product that tautomerizes to give two possible Δ1-imine species in which C-2 isomerizes between (R) and (S) configurations   by an activated and terminal Zn-coordinated hydroxide ion , 21, 22.Fig. 1products , but β-lproducts , 26, 27 products C. This mand MBLs  and alsoand MBLs . CarbapeFig. 1D) , 31, 32.Stenotrophomonas maltophilia  as an organism for which treatment strategies must be developed. Many L1 variants have now been identified in clinical S. maltophilia isolates, with sequence identities ranging as low as 76% to the originally characterized L1 from strain IID 1275 (in vitro but one (referred to as L1e) hydrolyzes nitrocefin and imipenem more poorly than the parent enzyme  face of the pyrroline/dihydrothiazine rings, suggesting that a common stereoisomer is favored for high-affinity interactions with L1. Furthermore, these data indicate that L1-catalyzed cephem hydrolysis can occur without either loss of the 3′ leaving group or protonation at N-5, expanding the range of mechanisms by which L1 inactivates diverse β-lactam antibiotics. These results can be exploited in developing antibiotics resistant to the action of dizinc MBLs, as well as potent MBL inhibitors, by exploring novel compounds that form long-lasting complexes.Understanding the interactions that stabilize hydrolyzed antibiotics in the active site of dizinc MBLs is key to developing novel antibiotics that are more poorly hydrolyzed by these enzymes as well as inhibitors that can exploit such interactions to generate long-lasting complexes. Here, we report the use of high-resolution X-ray crystallography to explore the interactions of six different hydrolyzed β-lactams, with the originally described, and most extensively characterized L1 enzyme that is a major contributor to S. maltophilia IID 1275 catalyzes the hydrolysis of a wide variety of bicyclic β-lactam antibiotics, including those from the carbapenem, penem, cephem, and penicillin classes (kcat/KM) greater than 0.8 s−1/μM−1  and collected X-ray diffraction data at resolutions extending to 1.43 to 1.63 Å . Analysi density . Experim2 side chains, The products derived from mecillinam and cefoxitin could both be modeled with two conformations, resulting from the flexibility of their C-6 or C-3 substituents -configuration. All of the respective C-2 substituents  as their Δand 1SML ) to 3.8/and 1SML B and S3.and 1SML ).Figure C and D), including with an L1 variant , as observed in complexes of hydrolyzed tebipenem, doripenem, and ertapenem (PDB 7O0O (6UAH (F), represented by L1:panipenem (this work), L1c:imipenem  with L10O (6UAH ), the Zn0O , the brTaken together, these results suggest that the precise binding mode of (hydrolyzed) carbapenems is determined by a combination of the presence/absence of the carbapenem 1β-methyl group and the presence of Tyr33 in L1. In particular, with the 1β-hydrogen carbapenems  interaction of the C-6 carboxylate with Tyr33 (binding mode 2) is made possible by a rotation (about the C-5 - C-6 bond) that would otherwise be sterically hindered by a β-methyl at C-1. As a consequence, 1β-methyl-carbapenems  adopt binding mode one in interactions with L1. The proposed role of Tyr33 is supported by comparisons with carbapenem complexes of the related subclass B3 MBL SMB-1 , which lB). However, a penicilloic N-formyl amide, previously identified as a minor rearranged degradation product of mecillinam breakdown in solution (A). To support our assignment of this species as the best explanation of our crystallographic data, the products of mecillinam degradation catalyzed by L1 in solution were investigated by NMR spectroscopy. A time series of one-dimensional 1H-NMR experiments identified the mecillinam hydrolysis product as slowly rearranging  to multiple species. Prolonged (2 week) incubation followed by semi-preparative HPLC enabled the isolation of the four major observed products (1), its non-enzymatically formed epimer (2), and both epimers of the non-enzymatically formed rearranged penicilloic N-formyl amide (3 and 4). Of these, the penicilloic N-formyl amide (4) is observed bound in the crystal structure and is the only product to satisfactorily fit the experimental electron density. These data also show that the rearranged penicilloic N-fromyl amide can form from the enzyme-catalyzed penicilloic acid. The crystallographically observed product (4) contains a newly formed N-formyl amide at C-6 that is positioned 3 Å from the Zn-bridging water/hydroxide . Thus, although no known penicillin G breakdown products seem to fit the electron density in L1c, it is possible that the observed product in PDB 6U0Z is a mixture of different species (resulting in poorly defined electron density) or a yet-to-be-identified penicillin-derived rearrangement product, rather than a penicilloic acid. We note that rearranged penam degradation products have not previously been observed crystallographically complexed with B1 MBLs, with the expected penicilloic acid (1) being well-defined as a complex with NDM-1 . Althou6U0Z B. Thus, th NDM-1 , 52.3 group, green in 3) and carboxylate groups (A). This binding mode causes a slight decrease in the distance between Asp120 and the Zn-bridging water from 2.8 Å  substituents.Both cefoxitin and cefmetazole contain a 7α-methoxy substituent (Re groups A. This bPDB 1SML ) to 2.6 ML (1SML ), consisML (1SML  and withML (2AIO ). The C-2-leaving group will lose this on hydrolysis by β-lactamases from both the SBL and the MBL classes, with subsequent formation of a C-3 = C-3′ exomethylene group (see C). However, in our studies, both cephem-derived hydrolysis products were modeled with the C-3′ leaving group intact as evidenced by the presence of electron density for the exocyclic sulfur (cefmetazole) or oxygen (cefoxitin) atoms of the C-3 (R2) substituents positioned outside the plane of the cephem dihydrothiazine ring. Further, electron density next to C-3, that would indicate the presence of a C-3 = C-3′ exomethylene group, is absent. Indeed, C-3 is clearly sp3 hybridized and protonated at the equivalent face of the dihydrothiazine ring as is C-2 in the pyrroline ring of carbapenem-derived Δ1-imine products (note that the (3R) cephem and (2S) carbapenem configurations are stereochemically equivalent). The tetrahedral nature of C-3 then dictates that the β-lactam-derived nitrogen (N-5) is present as the imine, rather than the enamine, tautomer. This suggests a mechanism of hydrolysis involving C-3 protonation by a non-activated water molecule in the active site on the β-face of the dihydrothiazine ring , which is similar to the protonation of C-2 on the equivalent face of the pyrroline ring during carbapenem hydrolysis (A).It is generally thought that cephems with an appropriate Rroup see C. Howevedrolysis A.Figure Our extensive crystallographic data, combined with previous studies, enable new insight into the chemistry and binding modes of hydrolyzed β-lactam antibiotics from all classes in the active site of L1. In particular, all of the observed complexes are stabilized by common interactions with a hydrophobic binding pocket and three conserved hydrogen-bonding residues . Moreover, by expanding our knowledge of L1:β-lactam interactions, our data reveal the extent to which these are replicated in the binding of inhibitors of different classes and go some way to rationalizing the differential activities of these toward L1.e.g., boronates, thiols) displace the Zn-bridging hydroxide  do not orbactam . Howeverorbactam , which morbactam . In mostorbactam ) geometrB). All of these binding modes feature coordination of the MBL Zn2 metal ion by a carboxylate, while the Zn-bridging hydroxide interacts with either the indole N of InCs, a phosphonate oxygen of PMPCs, or a hydroxyl group of cyclobutanones. The InCs are extremely potent inhibitors of L1 (as judged by in vitro activities), interacting with Ser221/Ser225 in the active site without bidentate coordination of Zn2 , and further clash, rather than make productive interactions, with L1 Tyr33  cannot rule out that these may result from rebinding events of hydrolyzed β-lactams, rather than representing states along the hydrolysis pathway. This is probably the case for the rearranged mecillinam degradation product, as we show that following initial hydrolysis its formation is likely non-enzymatic and possibly results from an intermolecular reaction of an intermediate with free hexamethyleneimine .S)-Δ1-imine tautomer. It has been suggested that diastereoselective protonation at C-2 of an anionic intermediate can occur on the enzyme to form the (S)-Δ1-imine in a mechanism for carbapenem hydrolysis by NDM-1 (A). Studies on the reactions of both MBLs and SBLs with the carbapenem biapenem also reveal that the (S)-imine is the major product formed (2-enamine can rapidly tautomerize (either on enzyme or in solution) to the (R)-Δ1-imine, which then converts slowly to the more stable (S)-Δ1-imine over longer time scales (R)-Δ1-imine, and note that its formation at the L1 active site is likely to be sterically unfavorable.The possibility of complex formation through rebinding is also important when considering the crystallographically observed hydrolyzed carbapenem complexes, all of which are in the  cephalosporin-derived products, protonated at the β-face of their dihydrothiazine ring, are stereochemically equivalent to the (2S) carbapenem-derived products, highlighting that configurations of antibiotic-derived products protonated on the opposite (α-) face are less preferred and less stable in the MBL active site. Further, our previous QM/MM simulations indicate that the Δ2-enamine product of ertapenem was less stable at the active site than the (S)-Δ1-imine (S)-Δ1-imine on the active site, as previously suggested  (S)-Δ1-imine and then rebinds or the (S)-Δ1-imine slowly accumulates in the active site, leaving more slowly than other tautomeric forms/stereoisomers  due to the increased stability of the L1 complex. Recent QM/MM studies  proceeds via a common anionic intermediate (in which N-5 is negatively charged) that is stabilized by interaction with Zn(II) (B). The resultant products differ in that the C-3′ group is either eliminated  involves protonation of the amide N of the anionic intermediate without tautomerization of the cephem dihydrothiazine (4ZJ3 (6Q5F (4X55 (2 group) (Cephem hydrolysis B proceedh Zn(II) , 64, 65.h Zn(II) B. The reiminated  or is red by NMR ). A third by NMR B involvethiazine . Crystalne (4ZJ3 ); and inJ3 (6Q5F  and PDB 5F (4X55 ), most c5F 4X55 , 66, 71 , 71 B pr5F  .B and 1SML  or thatand 1SML . In the ML (3SPU ) appear ML  formed prior to the loss of the C-3′ leaving group , wito NDM-1 . It was ng group . Howeverng group . A specing group .2 group at C-3 after a 90 min soak  hydrolysis by L1, which is 100-fold less efficient than that of the 7α-methoxy cephalosporins cefoxitin and cefmetazole, likely differs as a crystal structure shows loss of the Rmin soak , reflectdentical , regardlsp3 hybridized at C-3, that retain their C-3′ leaving groups, implies that for L1-catalyzed hydrolysis of at least some cephems, C-3 protonation of an anionic intermediate, via a non-activated water molecule in the active site, is preferred to N protonation by a Zn-bound water. It is then possible that a similar pathway may operate in carbapenem hydrolysis by L1 and other MBLs such as NDM-1. How the initial intact β-lactam binds  is yet to be established, with the crystal structures herein likely snapshots of hydrolysis endpoints. However, our results indicate that multiple β-lactam-derived hydrolysis and/or degradation products can bind to the L1 active site and importantly establish the most stable complexes and species. This information can be exploited to design compounds able to form long-lived species at the active site of dizinc MBLs such as L1, able to function either as small molecule MBL inhibitors or as novel antimicrobials resistant to MBL-catalyzed degradation. Such entities will be particularly important in counteracting AMR mediated by subclass B3 MBLs, such as L1, that currently evade the activity of the most potent, and closest to the clinic, MBL inhibitors.Our crystallographic results reveal that L1 manifests preferential binding of species formed after diastereoselective protonation, at C-2/C-3, on the β-faces of the pyrroline and dihydrothiazine rings of carbapenems and cephalosporins, respectively . Our obsS. maltophilia strain IID 1275 (4(SO4)2] in ChrysChem24 sitting drop plates (Hampton Research). Crystals grew within 3 weeks and were soaked by placing them into cryo-solution  supplemented with antibiotics from time points ranging from 30 min to 48 h before flash-cooling in liquid nitrogen. Data were collected and structures determined from crystals soaked in 18 mM tebipenem for 16 h, 12.5 mM doripenem for 16 h, 18 mM panipenem for 8 h, 10 mM mecillinam for 16 h, 15 mM cefoxitin for 23 h, and 20 mM cefmetazole for 22 h. Diffraction data were collected at Diamond Light Source on beamlines I24 (mecillinam) and I03 . Data were processed using the xia2 pipeline at Diamond Light Source . The following wavelengths and extinction coefficients were used: tebipenem ; panipeM−1 cm−1 ); doripeM−1 cm−1 ); mecillM−1 cm−1 ); imipenM−1 cm−1 ); meropeM−1 cm−1 ); biapenM−1 cm−1 ).2O or D2O, final concentration: one or 4 mM) was dissolved in 25 mM Tris-d11 in D2O, pD eight, or in 25 mM Tris-d11 in H2O, pH 7.5  D2O). Hydrolysis of mecillinam was achieved after the addition of L1 . NMR spectra were recorded immediately after the addition of the β-lactamase.Purified L1 was buffer exchanged into phosphate buffer  using microspin columns (BioRad) and concentrated using Amicon Ultra Centrifugal Filters . Experiments were performed in 5 mm regular NMR tubes at 298 K. Mecillinam  or a Bruker AVIII 750 MHz NMR spectrometer  equipped with an inverse 5 mm TCI 1H cryoprobe. Carr-Purcell-Meiboom-Gill (CPMG) NMR spectra were recorded by applying the PROJECT-CPMG sequence  and TopSpin 3.6.1 .NMR spectra were obtained using a Bruker AVIII HD 600 equipped with a BB-F/H Prodigy Nsequence . The exp11 in D2O, pD 8.0) and L1  were incubated for 2 weeks at room temperature. NMR spectra were recorded to measure the extent of the reaction. The products were isolated using a Shimadzu HPLC system, equipped with a SunFire semiprep column . Elution employed a gradient of acetonitrile in water : 0 to 12 min (0.5 %), 12 to 47.5 min (0.5–60%), 47.5 to 50 min (60%), 50 to 52 min (60–98%), 52 to 55 min (98%), 55 to 57 min (98–0.5%), and 57 to 60 min (0.5%). The isolated compounds were lyophilized to remove the solvent and dissolved in D2O for NMR analysis.Mecillinam , 7ZO3 (tebipenem soak), 7ZO4 (panipenem soak), 7ZO5 (mecillinam soak), 7ZO6 (cefoxitin soak) and 7ZO7 (cefmetazole soak).For all crystal structures presented herein, coordinates and structure factors have been deposited to the Worldwide PDB under accession codes This article contains supporting information , 60, 71.The authors declare that they have no conflicts of interest with the contents of this article.


Maintenance electroconvulsive therapy (mECT) is an option in the treatment of affective disorders which progress is not satisfactory. It is certainly neglected and underused during the clinical practice.To evaluate the efficacy of mECT in reducing recurrence and relapse in recurrent depression within a sample of three patients.We followed up these patients among two years since they received the first set of electroconvulsive sessions. We applied the Beck Depression Inventory (BDI) in the succesives consultations for evaluating the progress.The three patients were diagnosed with Recurrent Depressive Disorder (RDD). One of them is a 60 year old man that received initially a cycle of 12 sessions; since then he received 10 maintenance sessions. Other one is a 70 year old woman that received initially a cycle of 10 sessions; since then she received 6 maintenance sessions. The last one is a 55 year old woman that received initially a cycle of 14 sessions; since then she received 20 maintenance sessions.All of them showed a significant reduction in depressive symptoms evaluated through BDI and clinical examination. In the first case, we found a reduction in the BDI from the first consultation to the last that goes from 60 to 12 points; in the second case, from 58 to 8 points; and in the last case, from 55 to 10 points. The main sections that improved were emotional, physical and delusional.As side-effects of the treatment, we found anterograde amnesia, lack of concentration and loss of focus at all of them.We find mECT as a very useful treatment for resistant cases of affective disorders like RDD.It should be considered as a real therapeutic option when the first option drugs have been proved without success.None Declared


Terahertz steady-state and time-resolved conductivity and permittivity spectra were measured in 3D graphene networks assembled in free-standing covalently cross-linked graphene aerogels. Investigation of a transition between reduced-graphene oxide and graphene controlled by means of high-temperature annealing allowed us to elucidate the role of defects in the charge carrier transport in the materials. The THz spectra reveal increasing conductivity and decreasing permittivity with frequency. This contrasts with the Drude- or Lorentz-like conductivity typically observed in various 2D graphene samples, suggesting a significant contribution of a relaxational mechanism to the conductivity in 3D graphene percolated networks. The charge transport in the graphene aerogels exhibits an interplay between the carrier hopping among localized states and a Drude contribution of conduction-band carriers. Upon photoexcitation, carriers are injected into the conduction band and their dynamics reveals picosecond lifetime and femtosecond dephasing time. Our findings provide important insight into the charge transport in complex graphene structures. 3D Graphene aerogels add high porosity and elasticity to the optoelectronics of the graphene family materials. Their charge carrier transport is governed by an interplay of the hopping and Drude-like intraband conductivity. These highly-demanded properties open up new opportunities for graphene in various high-performance devices, such as fast field-effect transistors,7 far-infrared detectors,8,9 plasmonic devices,10 harmonic generators and frequency converters,11 optical electric field modulator.12 For such devices, it is of primary importance to accurately understand graphene's optical and electronic properties in the THz regime.Graphene has emerged as an outstanding two-dimensional (2D) material due to its unique optical and electronic properties such as high electron mobility,e02/4ħ, due to the interband carrier transitions.4,13 However, in the THz region, doped graphene rather exhibits a Drude conductivity behavior14–16 owing to dominating intraband transitions. A plasmonic conductivity peak is also frequently observed in the THz range for epitaxial graphene.14,15,17–19 In multilayer graphene, THz wave absorption strongly depends on the misorientation angles and stacking arrangements between the graphene layers;20 the Drude conductivity then leads to a decrease in THz transmission in multilayer systems.21 Recently, a conductivity peak due to van Hove singularity at 2.7 THz on top of a Drude-like response observed in bilayer graphene was attributed to commensurate twisting of graphene layers.22 Briefly, multiple physical phenomena can modulate the electronic coupling between graphene layers, suggesting exciting new opportunities for tuning the THz response in more complex three-dimensional (3D) graphene structures. In contrast to extensive efforts on the graphene in a 2D form, optical studies of 3D graphene structures in the THz range are still limited23,24 and a deeper insight into the charge transport has not been reported yet.Single-layer graphene exhibits optical absorption in the visible to mid-IR range close to the well-known theoretical value of 25,26 Compared with traditional 2D graphenes, GAs provide high porosity and large effective surface area; GAs are also lightweight, elastic, highly conductive, and mechanically robust, making them promising for various applications, e.g., strain and pressure sensing.27 However, THz optoelectronic properties of GAs have not been explored in detail yet.In recent years, thanks to the development of graphene synthesis, 3D graphene networks assembled in free-standing covalently cross-linked graphene aerogel (GA) structures that maintain the characteristic properties of single-layer/multilayer graphene have been realized.14–19,28 We investigate the role of defects in GA on the character of the charge carrier transport by controlling the transition between reduced-graphene oxide and graphene using high-temperature annealing. Ultrafast THz photoconductivity measurements in high-temperature annealed samples reveal a photoinduced Drude-like absorption process with a picosecond characteristic decay.In this work, we report experimental observations of the THz conductivity and permittivity in graphene assembled in free-standing covalently cross-linked GA. The experiments reveal an interplay between the Drude and hopping conductivity, which is significantly different from the transport observed in 2D graphene layers.−1 mbar) for 16 h to get a cylindrical sample of stable reduced graphene oxide (rGO) aerogel. The obtained rGO aerogel was kept at room temperature 25 °C and subsequently annealed in vacuum (2.3 × 10−4 mbar) at temperatures of 400, 750, 1300 and 2700 °C for 30 minutes. The samples were prepared with the initial thickness of 3–4 mm and their dc conductivity was determined at this stage. In order to obtain at least partially transparent samples for THz experiments, they were subsequently thinned down to various thicknesses .A scheme of the fabrication process is depicted in Scanning electron microscopy (TESCAN MAIA3) was used to characterize the morphology of the samples. Furthermore, X-ray photoelectron spectroscopy  was employed to check the elemental composition and Raman spectroscopy (Renishaw inVia setup using a 442 nm laser) was used to assess the crystallinity and crystallite size of the samples. Dc conductivity measurements were performed by linear four-point probe method by slightly pressing the sample clamped between two glass slides on the top of predeposited electrodes (≤5% strain) to achieve a good electrical contact.29 was used to measure the THz waveforms transmitted through the samples. The complex refractive index N of the samples was calculated from the transmission spectra including the Gouy shift correction.29 Subsequently, spectra of the complex permittivity ε = N2 and conductivity σ = −iωε0(ε − 1) were evaluated. In this work, we display real parts of ε and σ because they show the most pertinent spectral features. Low-temperature measurements were performed in an optical cryostat  with mylar windows. The thicknesses of the samples were measured separately using an Olympus optical microscope and used for the determination of their THz refractive indices.30Femtosecond oscillator-based time-domain THz spectroscopy setup (using a photo-switch emitter TeraSED and an electro-optic gated detection scheme with 1 mm thick ZnTe)α of GA was larger than 25 mm−1. In this case, it can be shown31 that the THz photoconductivity spectrum (at a pump-probe delay τp) can be evaluated using the following formula:σnorm is the photoconductivity of the aerogel sample per single unit charge, ΔE is the measured transient  transmitted THz spectrum upon the sample photoexcitation with a photon fluence ϕ, E is the reference transmitted THz spectrum (without photoexcitation), z0 is the vacuum wave impedance, e0 is the unit charge, and N is the measured complex refractive index of the GA sample in equilibrium. The formula is deduced from the case (B1) of The optical pump – THz probe experiments in a collinear arrangement were carried out at room temperature using a Ti:sapphire ultrafast amplified laser system . The laser fundamental wavelength was used for the sample photoexcitation and also for the generation and electro-optic gated detection of the THz probing pulse in a pair of 1 mm thick (110) oriented ZnTe crystals. The sample was attached to a metallic aperture with an inner diameter of 3 mm. The pump beam was spatially expanded in order to excite the exposed part of the sample quasi-homogeneously. Samples thinned to ∼100 μm were completely opaque for the pump beam. This means that the effective optical absorption coefficient 25Using the hydrothermal synthesis followed by freeze-drying and high-temperature annealing, we “engineered” cross-linked graphene networks in GA with the same morphology but with different amounts of structural defects. The morphology and Raman spectroscopy analysis of the GA samples are depicted in −1, corresponding to the D and G bands of rGO. In contrast, GA samples annealed at 2700 °C depict an additional 2D peak at 2720 cm−1 and a significant reduction in the D peak intensity. This demonstrates that the annealing was efficient in improving the crystallinity and removing oxygen functional groups from the GA, facilitating a transition from rGO to graphene.Raman spectra of the non-annealed samples show two intense peaks at 1369 and 1596 cmLa) and the density of defects (nD) in the samples from the Raman peak intensity ratio I(D)/I(G),32,33 see La and nD upon annealing up to 750 °C. However, as the annealing temperature is raised to 1300 °C, a clear increase in the crystallite size along with the reduction of the defect concentration occurs. At this temperature, most of the oxygen functional groups are unstable and desorb from the rGO,34–37 as corroborated with the XPS compositional analysis in La and nD by a factor of ∼6 compared to the non-annealed GA.We evaluated the crystallite domain size  hopping conductivity contribution. The Drude term is considered frequency independent in the accessible spectral range because the carrier dephasing time is very short, as confirmed below by the time-resolved THz measurements. The dispersion of the real part of the permittivity can be simply deduced:We propose to describe the observed processes by the random free-energy barrier model proposed by Dyre.ε∞ and the upper cut-off frequency ωmax do not show a clear trend with annealing temperature and are not important for further discussion of the physics of our materials. On the other hand, the individual contributions to the conductivity σDrude and σhop are fundamental; their behavior can be clearly seen in versus annealing temperature as the Drude conductivity term obtained from the THz data, but their absolute values are systematically different (by about a factor of 3). The applied stress in dc measurements might have caused some change in the conductivity due to the induced strain in the samples,25,26 but it could not lead to a 3-fold increase. Note, however, that a precise quantitative comparison between the dc and THz conductivity is generally difficult in complex materials since different areas and conducting pathways are probed by the two techniques.Note that the Drude term does not contribute to the real part of the permittivity in the case of the very short carrier scattering time: the whole observed dispersion is due to the relaxation mechanism (hopping). The fits of the experimental data using the model  are shown in E, measured at a pump-probe delay of 2 ps) and the equilibrium transmission waveform (E) are shown in the inset of E has an opposite sign than E). The kinetics in 44The optical pump-THz probe measurements of GA samples exhibit very fast kinetics . The lifetime of photocarriers in these low-temperature annealed samples is also somewhat shorter, ≲0.4 ps, and it is at the limit of the resolution of our experimental technique. This acceleration of the charge dynamics naturally corroborates a larger concentration of defects in these GA samples due to significantly more traps for the photocarriers.The THz photoconductivity spectra evaluated following 2 V−1 s−1 for the GA sample annealed at 2700 °C) can then be understood as an order of magnitude estimate  of the charge carrier mobility in this sample. Assuming similar carrier mobility in the ground state and using the steady-state Drude conductivity value  using the ratio between the mass density of our GA samples (6–7mg cm−3) and pristine graphene (2.3 g cm−3).45 It should be noted that the mobility is very low compared to standard 2D graphene layers,46 suggesting that there is ample space for improvement.If we assume that each absorbed pump photon generates a mobile charge, then the initial normalized transient conductivity directly represents the mobility of the photogenerated charges. The maximum value of the dynamics in 31 The character of the measured spectra indicates that the investigated graphene sheets form a network with a high degree of percolation.The measured steady-state and transient effective conductivity spectra reflect the morphology and particularly the percolation degree of materials.We also investigated the steady-state spectra of the samples as a function of temperature. 13 cm−2 while their mobility is very low (of the order of 10 cm2 V−1 s−1), thus indicating a large space for improvement. Optical pump-THz probe results reveal Drude photoconductivity with very fast decay (∼1 ps). Our study opens new prospects for understanding charge transport in multidimensional structures made up of 2D layers.This work represents a systematic investigation of the charge carrier dynamics in the 3D structure of graphene aerogels. The charge transport mechanism exhibits both intraband transitions and hopping transport modes at THz frequencies. This behavior is strikingly different from that observed in 2D graphene. The hopping process controls the transport in defect-rich parts of the 3D graphene networks. Conversely, in parts with low defect concentration, a Drude band-like conductivity with very short carrier scattering time takes over the transport mechanism. Higher annealing temperatures lead to an increase in the Drude conductivity and, to a lesser extent, in the high-frequency hopping contribution due to the presence of shallow states. Free carrier density in the sample annealed at 2700 °C reaches 10The authors declare no conflict of interest.