Graphene has become an ideal reinforcement for reinforced metal matrix composites due to its excellent mechanical properties. However, the theory of graphene reinforcement in graphene/aluminum matrix composites is not yet well developed. In this paper, the effect of different temperatures on the mechanical properties of the metal matrix is investigated using a classical molecular dynamics approach, and the effects of the configuration and distribution of graphene in the metal matrix on the mechanical properties of the composites are also described in detail. It is shown that in the case of a monolayer graphene-reinforced aluminum matrix, the simulated stretching process does not break the graphene as the strain increases, but rather, the graphene and the aluminum matrix have a shearing behavior, and thus, the graphene “pulls out" from the aluminum matrix. In the parallel stretching direction, the tensile stress tends to increase with the increase of the graphene area ratio. In the vertical stretching direction, the tensile stress tends to decrease as the percentage of graphene area increases. In the parallel stretching direction, the tensile stress of the system tends to decrease as the angle between graphene and the stretching direction increases. It is important to investigate the effect of a different graphene distribution in the aluminum matrix on the mechanical properties of the composites for the design of high-strength graphene/metal matrix composites. As a two-dimensional carbon nanomaterial, graphene has extremely excellent mechanical, optical, and electro-catalytic properties ,2 and hiMetal matrix composites are widely used in aerospace and automotive industries for their excellent properties such as high strength-to-weight ratio and low coefficient of thermal expansion. In recent years, fruitful results have been achieved by using nano-phase reinforced metal matrix composites such as carbon nanotubes and graphene ,13, whicIt is well known that direct experimental observation of the details and patterns of structural evolution during material deformation is costly and can be limited by modern techniques such as electron and optical microscopy, so new avenues have been explored. Due to the rapid development of computer technology, molecular dynamics (MD) simulations are playing an increasingly important role in the design and development of new materials ,24,25,26Molecular dynamics is a set of molecular simulation methods that rely on Newtonian mechanics to simulate the motion of a molecular system under certain interaction laws and computers to calculate the phase orbit of a collection of particles to determine the static and dynamic properties of the system, i.e., the thermodynamic and other macroscopic properties of the system. Molecular dynamics simulations were performed using Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) . The Al is paper . The modis paper ; OVITO iFirst, we take a perfect aluminum structure as the object of study and systematically investigate its mechanical properties at different temperatures. As shown in As shown in As shown in Before studying the composite properties, we investigate the mechanical behavior of perfect graphene. As shown in As shown in After discussing the mechanical properties of perfect graphene and the aluminum matrix, we investigate the mechanical properties and failure behavior of perfect graphene/aluminum composites using molecular dynamics. In our previous work , we founFrom As shown in As shown in From The effects of temperature and graphene on the mechanical properties of an aluminum matrix were investigated by molecular dynamics methods. The stress in the aluminum matrix decreased with increasing temperature, and a similar trend was observed for the modulus. In the study of monolayer graphene-reinforced aluminum substrates, it was found that holes first appeared at the graphene–substrate interface. As the strain increases, the holes expand, and further expansion of the crack leads to cracking of the substrate, with graphene as the “bridge” connection and stress concentrated on the “bridge” of graphene. The study of the three-layer graphene-reinforced aluminum substrate found that the shear between the outer graphene layer and the substrate occurs late in the strain, which is the main factor for stress retention in the material. In studying the effect of graphene size and angle on the mechanical behavior of graphene/Al composites, we found that in the parallel x-direction, the tensile stress tends to increase with increasing percentage of graphene area. In the perpendicular x-direction, the tensile stress tends to decrease with the increase of the percentage of graphene area. In the angle with the x-axis, the tensile stress tends to decrease with increasing the angle of graphene with the x-axis. By investigating the effects of temperature and graphene on the mechanical properties of an aluminum matrix, it provides some theoretical references for the study of graphene-reinforced metal matrix composites experimentally.


In 2018 we published the James Lind Alliance (JLA) top 10 priorities for clinical research in cystic fibrosis (CF), chosen jointly by the patient and clinical communities. These priorities have led to new research funding. To establish whether priorities have changed with novel modulator therapies, we undertook an online international update through a series of surveys and a workshop. Patients and clinicians (n=1417) chose the refreshed top 10 from 971 new research questions (suggested by patients and clinicians) and 15 questions from 2018. We are working with the international community to promote research based on these refreshed top 10 priorities. In 2018 we published the top ten research priorities for cystic fibrosis (CF), jointly developed by the CF patient and clinical communities.The original top ten priorities were produced by working with the NIHR-supported James Lind Alliance (JLA) who facilitate priority setting partnerships (PSPs) using methodology which brings patients, carers and clinicians together to prioritise research questions. We were the first PSP to undertake a refresh of disease-specific research priorities and have worked with the support of the JLA Lab – an exploratory space that supports innovation.Our PSP refresh took place October 2021 – November 2022. We published our protocol before commencing.From January to February 2022, we conducted the first of two online surveys (Survey Monkey). The elicitation survey requested respondent demographics and asked participants to select their top three priorities from the previous top twenty questions. Respondents were also asked to submit up to two new priorities. Surveys were advertised through professional and charity groups and our bespoke Twitter account (@questionCF). Both surveys were available in multiple languages. In a change to our original PSP, non-industry researchers were also invited to participate in the refresh.Two researchers (NR and SS) independently reviewed all submissions with adjudication by the steering and management groups. Responses which did not include a question and out of scope questions were removed. Where several questions addressed the same issue, they were consolidated into a single “umbrella question”. Any question already answered in the scientific literature was removed.The standardised umbrella questions were then taken forward to the prioritisation survey (September – October 2022). This allowed respondents to rank their top ten questions. Cumulative scores were calculated for each question and the questions ranked in priority order for the following four groups:pwCFThe subgroup of pwCF who currently do not have access to CFTR modulatorsFriends and family of pwCFHealthcare professionals and researchersQuestions in the top ten priorities for each of the three main groups, plus the top five for the sub-group of pwCF not on CFTR modulators , were th10.1136/thorax-2023-220100.supp1Supplementary dataWe had 1608 responses from 1370 respondents to the elicitation survey (59 responses were ‘empty’ and 179 respondents completed the survey more than once). An additional 971 questions were submitted. This is the first refresh exercise to update the JLA top 10 priorities for a specific medical condition. Our methodology ensured equal participation from people with CF, their family and friends and the clinical/research communities. We have produced a refreshed top 10 list of research questions, which should guide researchers and funders. Four priorities were identified in the original PSP and remain priorities. Six priorities were newly identified by the PSP refresh.A strength of our study is its international reach. We had a large number of respondents, from countries across the world. While CF is predominantly a disease of Northern European descent, recent prevalence studies show an increase in countries where it was not previously reported.With the arrival of transformational therapies, available to most  pwCF, it is vital that the research priorities of funders and researchers remain aligned with those of the patient and clinical communities. Publication of the previous JLA top 10 priorities for CF has led to international funding  for many clinical studies designed to address these priorities.The previous PSP led directly to the CF-STORM trial  which was developed in response to Question 1 (“What are the effective ways of simplifying the treatment burden of people with Cystic Fibrosis?”) and to the GIFT-CF research programme


The porous interconnected framework and high crystallinity of 3DG could contribute to not only its facilely mixing with epoxy resin, but also its effective incorporation into the conductive network of zinc micro-flakes, thus enhancing the corrosion resistance of its ZRE coating at a lower content. The innovative technology to improve the anti-corrosion performance of the ZRE coatings via using the 3D graphene fillers should be capable to be extended to other 2D fillers, such as MXenes. To improve the corrosion inhibition of zinc-rich epoxy (ZRE) composite coatings and shed light on the influence of the spatial structure of graphene fillers on the coatings’ performance, three-dimensional graphene (3DG) and a conventional graphene sheet (G) were used to modify the ZRE composite paint, respectively. The effect of introducing the 2D G fillers on the anti-corrosion behavior of ZRE was studied comprehensively, and its optimal content was determined to be 0.5 wt%. Interestingly, it was found that, comparing with 2D graphene sheets, the corrosion resistance of the ZRE coating could be enhanced more significantly with incorporating even less 3DG. With introducing only 0.1 wt% 3DG, the corrosion current intensity of the resulting 3DG/ZRE coating was reduced to be about 1/10 that of the G/ZRE coating with the same graphene content and 27% of that of the optimized G/ZRE. The corrosion products of the coating were analyzed with the XRD technique. The results indicated that, in contrast to neat ZRE coating, Zn Recently, two-dimensional 2D) multi-functional materials, including graphene, graphitic carbon nitride, and MXene, have aroused extensive research interest all over the world, and a large amount of work on their facile fabrication and applications in many fields ,2,3,4. CD multi-fDue to their high aspect ratio and 2D structure, graphene sheets are ready to aggregate in the polymer matrix, resulting in a great challenge to prepare their composite with a high performance. To alleviate this, graphene derivatives, including graphene oxide or fluorinated reduced graphene oxide, have been developed and used to prepare the composites ,21,22,23Graphene foam has been developed in recent years. The most common method to prepare the foam is the so-called chemical vapor deposition method. With this method, the porous structure of graphene is controlled by the metal catalytic foam templates, and the thickness of the graphene foam strut-wall can be controlled to be within nanometers. Due to their unique porous 3D structures, their polymer composites with various advanced properties ,25,26, iIn this work, the influence of the spatial structure of high-crystalline graphene fillers on the corrosion resistance of ZRE coatings was studied. Besides conventional 2D graphene sheets, 3DG with a porous structure and interconnected graphene framework has been applied to modify the ZRE paint. Both the morphology and physical chemical properties of the as-prepared 3DG will be characterized. To avoid the influence of the crystalline structure of graphene on the anti-corrosive behavior of the modified ZRE coating, 3DG with a similar crystalline structure to graphene sheets has been used in this study. The anti-corrosion behaviors of the ZRE modified with different graphenes in the artificial simulated seawater (3.5 wt% NaCl solution) will be studied by the potentiodynamic polarization test and electrochemical impedance spectroscopy, as well as the long-term immersion test.Commercial graphene sheets  were purchased from Suzhou Tanfeng Technology Co. Ltd. . Epoxy (E-44) was provided by Xiya Reagent Co. . Xylene, n-butanol, and polyamide 651 were all chemically pure and purchased from McLean Group.  Commercial zinc flakes with a lateral size of 5–15 µm were ordered from Xiamen Empire New Material Technology Co. Ltd.  and a Q235 steel plates with a size of 30 mm × 40 mm were provided by Fuquan Metal Co. Ltd. .The whole process was illustrated as The crystalline structures of the graphene samples were characterized with an X-ray diffraction analyzer . The carbon structure of 3DG was analyzed with a Raman spectrometer . The morphology of the carbon sample was observed with a scanning electron microscope . An optical microscope (DM3000) was also utilized to observe the morphology of graphene fillers in epoxy resin.−1 and its potential range was set to be ±250 mV around the OCP. Three potentiodynamic polarization tests were carried out for both neat ZRE and 3DG/ZRE samples and the typical ones are reported in the work. The electrochemical impedance spectroscopy (EIS) tests were performed in a frequency range of 10 kHz~0.1 Hz at room temperature. The simulated seawater (3.5 wt% NaCl solution) was used as the corrosive electrolyte. The corrosion measurements of the ZRE coatings were measured on a CHI 760e electrochemical analyzer. A three-electrode half-cell system was constructed with a saturated calomel electrode (SCE) as the reference electrode, platinum plate as the auxiliary electrode, and ZRE coated Q235 plates as the working electrode. First of all, an open circuit potential test (OCP) of the sample was performed to ensure it reached a relative steady state. The potentiodynamic polarization test was carried out at a scan rate of 10 mV sDifferent from the common 2D morphology of normal graphenes a, 3DG isUnder the magnetic stirring in epoxy resin, the graphene framework monolith infiltrated with epoxy resin will be in situ broken into small fragment particles, whose morphology will have 3D irregular characteristics with a size larger than that of the conventional 2D graphene sheets . As show−1 and the 2D band at 2700 cm−1, together with a very weak peak for the D band around 1357 cm−1, could be observed in the Raman spectrum of 3DG  of 3DG is 0.66, which is much less than 1, implying its multi-layered structure [Similar to the research on the graphene deposited on catalytic metal substrates in the literature ,28, two m of 3DG . As the tructure .−7 A cm−2), which means a ~10-fold improvement in the corrosion resistance, comparing with that of neat ZRE (6.1 × 10−6 A cm−2). However, when further increasing the G content to 0.7 wt.%, the Icorr of its ZRE coating increases  of the ZRE coating could be used as a factor to evaluate their protection performance . Figure 0.01Hz of the ZRE coatings on the time is shown in 0.01Hz of all the coatings decreased quickly with the immersion time initially. Obviously, 0.5 wt% G/ZRE exhibited a larger |Z|0.01Hz than neat ZRE during the whole immersion test, confirming the positive effect of incorporating G on promoting the corrosion inhibition of ZRE coating. Additionally, the curve of 0.1 wt% 3DG/ZRE is always higher than that of 0.5 wt% G/ZRE all through the immersion process, demonstrating the superior effect of 3DG over the 2D graphene on enhancing the corrosion resistance of ZRE coating. The digital image of the exposed circle area of neat ZRE after the immersion test indicates that its major part is brown, together with some white areas  is nearly 1/10 that of 0.5 wt% G/ZRE (4.02 × 10−6 A cm−2) (The dependence of |Z| A cm−2) . Additio5 Ω·cm−2) was ~46 times that of 0.5 wt% G/ZRE (1.29 × 104 Ω·cm−2), which agreed well with the aforementioned Tafel test results (4 Ω·cm−2) was ~10 times that of 0.5 wt% G/ZRE (3.53 × 103 Ω·cm−2) (The Nyquist plots of the ZRE coatings after being soaked in simulated seawater are presented in  results . This im Ω·cm−2) , indicat2 products (Equations (1)–(5)), the insoluble complexes, including Willemite (Equations (6)) and hydrozincite (Equations (7)–(9)), could be also formed under certain conditions . After the continuous 300 h immersion test, the Q235 steel plates coated with either neat ZRE or 3DG/ZRE were taken out from the simulated seawater and their XRD patterns were taken to characterize the corrosion products. Consistent with the zinc corrosion products reported previously [5(OH)8Cl2H2O, and Zn5(CO3)2(OH)6, respectively. Interestingly, different from the presence of both complicated complex products on the soaked neat ZRE coating, no peak contributed from hydrozincite Zn5(CO3)2(OH)6 could be found on the pattern of the 3DG/ZRE coating after the 300 h immersion test. This could be due to the excellent barrier property of 3DG particles to carbon dioxide, demonstrating its great contribution to the surface protection of the ZRE coating.As suggested in the literature ,35, the eviously ,37, many5(OH)8Cl2H2O, as mentioned above. In contrast, a more significant morphology change occurred for the neat ZRE-coated sample after the immersion test, as indicated in As shown in (1)The corrosion resistance of the ZRE coating could be improved by incorporating common 2D graphene, and the optimal G content is 0.5 wt%.(2)The corrosion resistance of the ZRE coating could be further enhanced significantly by incorporating only 0.1 wt% 3DG.(3)Long-term immersion tests confirmed the superior effect of 3DG on improving the surface protection performance of ZRE as well.In this work, conventional 2D graphene sheets and 3DG fillers were used to modify the ZRE composite to improve the corrosion resistance of the ZRE coatings. These main conclusions are drawn:This work revealed, preliminarily, the significant effect of 3D graphene fillers on the surface protection performance of ZRE coating. However, to understand the spatial structure of graphene fillers on the properties of their ZRE coatings more comprehensively, further work needs to be done, such as the mathematical simulation of the contribution of 3DG to the cathodic protection process of ZRE coating.


Thus, these aberrant splicing events make promising novel therapeutic targets to restore functional gene expression. Moreover, presence of these cryptic exons is highly specific to patients and areas of the brain affected by TDP-43 proteinopathy, offering the potential to develop biomarkers for early detection and stratification of patients. In summary, the discovery of cryptic exons gives hope for novel diagnostics and therapeutics on the horizon for TDP-43 proteinopathies.TDP-43 is an RNA-binding protein with a crucial nuclear role in splicing, and mislocalises from the nucleus to the cytoplasm in a range of neurodegenerative disorders. TDP-43 proteinopathy spans a spectrum of incurable, heterogeneous, and increasingly prevalent neurodegenerative diseases, including the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum and a significant fraction of Alzheimer’s disease. There are currently no directed disease-modifying therapies for TDP-43 proteinopathies, and no way to distinguish who is affected before death. It is now clear that TDP-43 proteinopathy leads to a number of molecular changes, including the de-repression and inclusion of cryptic exons. Importantly, some of these cryptic exons lead to the loss of crucial neuronal proteins and have been shown to be key pathogenic players in disease pathogenesis (e.g. TDP-43 proteinopathy encompasses a wide spectrum of progressive neurodegenerative diseases and phenotypes. It is a key feature in ~ 97% of people with amyotrophic lateral sclerosis (ALS) and ~ 45% of people with frontotemporal dementia (FTD), a seminal discovery made in 2006 , 2. SubsTARDBP, the gene which encodes TDP-43, are rare, occurring in < 1% of ALS cases, but underscore TDP-43’s central role in disease pathogenesis [Common to TDP-43 proteinopathies is a lack of directed disease-modifying therapies, and an inability to confirm and stratify individuals that have TDP-43 pathology ante-mortem. The disease that is most frequently characterised by TDP-43 mislocalisation is ALS, which is a rapidly progressive paralysing illness with eventual death on average 3 years from symptom onset . Up to 1ogenesis –14. AlteIn this review, we will focus on a key ‘on-off’ consequence of nuclear TDP-43 loss of function, which has emerged over recent years – the occurrence of splicing defects leading to the erroneous inclusion of intronic sequences into mature mRNA, forming so-called ‘cryptic exons’ . With a CFTR gene, first realising TDP-43’s role as an RNA-binding protein and regulator of alternative splicing [POLDIP3, SORT1, and PFKP have all been used as reliable readouts for nuclear TDP-43 function in models with TDP-43 knockout, overexpression, and for studying TDP-43 ALS-causing mutations [TDP-43 was identified in 1995 as a suppressor of HIV gene expression. It was later discovered that TDP-43 expression promotes skipping of exon 9 in the splicing . Since tutations –27.TDP-43’s consensus binding motif comprises “UG”-rich dinucleotide repeats. High-throughput sequencing, combined with cross-linking immunoprecipitation experiments to discover direct TDP-43 mRNA targets, revealed that the majority of TDP-43’s binding sites in pre-RNAs were in introns , 27. A nWhat are the possible consequences that result from the insertion of novel non-conserved sequences in mRNA Fig. ? Most crSTMN2 gene [. This gene encodes for stathmin-2, a microtubule-associated protein needed for axonal growth and repair in neurons. Occurring after exon 1 and containing a stop codon and polyadenylation site, the cryptic exon leads to premature transcript termination and predicted formation of a truncated non-functional protein. Using in vitro neuronal models of TDP-43 depletion via knockdown, induction of TDP-43 mislocalisation, and pathological loss of function TDP-43 mutations known to cause ALS, the research groups demonstrated a resultant dramatic reduction in levels of mature STMN2 mRNA and functional protein. Crucially, the STMN2 cryptic exon was confirmed to be detectable also in ALS-FTD brains and spinal cords where TDP-43 pathology is present and specifically in patient post-mortem neurons with TDP-43 pathology isolated by laser capture or nuclear sorting [In 2019, a breakthrough discovery from two independent groups found that TDP-43 loss of function leads to the inclusion of a cryptic exon within the MN2 gene , 30. Thi sorting , 37. WitUNC13A is one of the top genetic risk factors for ALS, first reported in a genome-wide association study in 2009 [Munc13–1 in mice) have synaptic impairments and die soon after birth [UNC13A result in fatal microcephaly, cortical hyperexcitability, and myasthenia [UNC13A have been shown to increase risk of ALS and also shorten survival in patients. However, the underlying mechanism by which these UNC13A variants increase ALS risk was not known until earlier this year when, along with the Gitler and Petrucelli groups, we independently found that TDP-43 depletion resulted in inclusion of a cryptic exon between exons 20 and 21 of UNC13A, leading to reduced expression. Similarly to the STMN2 cryptic exon, this event can also be detected in post-mortem brain and spinal cord tissue from patients with ALS-FTD, exclusively where TDP-43 pathology is present, but not in healthy controls. in 2009 . UNC13A er birth . In humaasthenia . Single UNC13A SNPs, rs12608932 (A > C) and rs12973192 (C > G), were found to be located within the intron containing the cryptic exon, with the former SNP being located within the cryptic exon region itself. Especially given the large size of the UNC13A gene (~ 87 kb), the proximity of the SNPs to the cryptic exon led us to hypothesise a direct link between the SNPs and UNC13A cryptic splicing. Given that the cryptic exon is not present in RNA-sequencing data from healthy controls, even when homozygous for the risk SNPs (approximately 10% of Caucasians are homozygous for both risk SNPs), they seem not to be sufficient to cause cryptic exon inclusion on their own. Thus, it was hypothesised that rather than increasing ALS or FTD risk directly, the SNPs act by potentiating the expression of a cryptic exon which is dependent on TDP-43 depletion.Intriguingly, two of the associated UNC13A cryptic exon-containing transcripts in a dose-responsive manner compared to heterozygotes and non-carriers. This discovery provides a biological explanation for how presence of the UNC13A risk SNPs in ALS-FTD patients with TDP-43 pathology modifies disease outcomes, negatively affecting survival from symptom onset [Indeed, the risk SNPs enhance cryptic splicing and alter direct TDP-43 binding, thereby reducing its ability to physiologically repress cryptic exon inclusion. In keeping with this, patients homozygous for either SNP had more om onset , 41–47. ECD [Biomarkers for early diagnosis, disease stratification of subtypes and clinical stages, prognostication, and monitoring treatment response are vitally important, both for clinical trials and patient care. Fluid biomarkers of general neurodegeneration exist, most notably serum neurofilament light chain, cerebrospinal fluid (CSF) phosphorylated neurofilament heavy chain, and urinary p75ECD –50. HoweSTMN2 cryptic exon in post-mortem frontal cortex samples, in which its presence was able to discriminate between patients with FTD-TDP, progressive supranuclear palsy, and healthy control participants [Unlike canonical TDP-43 regulated splicing, the ‘on-off’ expression of cryptic exons only under TDP-43 nuclear loss of function makes them potential potent biomarker candidates for TDP-43 pathology. This proof of concept was first shown with the icipants .A key future direction will be to exploit this characteristic to develop methods that detect TDP-43 pathology ante-mortem in patient biofluids, such as in blood, CSF, urine, or tissue. Assays could involve either direct RNA detection of the cryptic exon itself, or detection of novel cryptic peptides for transcripts that escape NMD. An ideal biomarker would have a reproducibly high and linear correlation with disease activity, high sensitivity and specificity, stability throughout the day, and easy access to obtain a sample .UNC13A, undergo NMD and are therefore not stable. Moreover, if transcripts are at low levels, highly sensitive tools would be required. Detection of a panel of relevant cryptic exons deemed to be pathogenic players could theoretically increase sensitivity.With regards to biomarker detection of RNA, methods have been developed that can sensitively detect circulating RNA in the blood of individuals with Alzheimer’s disease that reflect disease-specific neural transcriptome changes . HoweverSTMN2, even though the cryptic-containing transcript is highly expressed and escapes NMD, attempts to detect the putative 16 amino acid cryptic peptide have not yet been successful. However, antibody generation to specifically detect pathological proteins is evolving, including in the field of ALS-FTD, with antibodies now available to detect dipeptide repeats that occur in the C9orf72 subtype of ALS-FTD. These have not only furthered our understanding of disease mechanisms, but are also being utilised to assess therapies [For cryptic exons that produce cryptic peptides, specific antibodies could be designed to detect these via immunohistochemistry, or ELISA and SIMOA-based assays. This has its own challenges, whether it be due to NMD of cryptic-containing transcripts, low expression, or instability of the peptides. For herapies –56.HDGFL2, detects the cryptic peptide in C9orf72-associated ALS. Strikingly, this includes pre-symptomatic mutation carriers [Excitingly, two new pre-print publications this year have independently reported the detection of cryptic peptides in the CSF of patients with ALS , 58. Irwcarriers . In SeddNeurofascin in these cells, highlighting how, if certain TDP-43 cryptic exons are expressed only by given cells, they could allow tracing of TDP-43 proteinopathy. Furthermore, recent work showing that pTDP-43 and loss of nuclear TDP-43 can be detected years before ALS disease presentation in central nervous system and non-central nervous system tissue suggests that detection of TDP-43 cryptic exons could possibly pre-date disease onset [Given the cell-type specificity of TDP-43 controlled cryptic exons, it is also possible that biomarkers towards cryptic exons could be designed to assess the flow of TDP-43 proteinopathy across the nervous tissue , 59, 60.se onset , 65.Another interesting avenue is the use of model systems to determine the timing of the emergence of cryptic exon inclusion events. For instance, studies assessing the presence of cryptic exons at different levels of TDP-43 knockdown in vitro, or in patients with or post-mortem tissue from varying clinical stages of ALS-FTD, could allow curation of a database reflecting which cryptic exons appear early and late in disease. Further analysis of this could provide a greater mechanistic understanding of cryptic exon biology and what factors make certain genes more sensitive and susceptible to the inclusion of cryptic exons with TDP-43 loss. Such a stratification may also have implications for biomarker studies and early diagnosis and stratification of people with ALS-FTD.Further work is necessary to utilise the specificity of cryptic exons to develop appropriate biomarkers, which could be transformative to the field of ALS-FTD and other TDP-43 proteinopathies. Particularly with new therapies on the horizon for neurodegenerative diseases, early diagnosis of individuals who are or will become affected and a readout for treatment response will be critical in halting the disease process at an early enough stage to pre-empt the point of no return in the ALS disease cascade .The discovery that certain cryptic exons, and potentially more that remain to be explored, underpin the disease process in ALS – either by impairing neuronal health, increasing ALS risk, or worsening disease progression – makes them exciting novel therapeutic targets. If early diagnosis can be achieved, the hope would be to restore expression of critical proteins to normal functional levels, or use splicing therapies to modulate and prevent aberrant splicing from occurring altogether in TDP-43 proteinopathies.STMN2 cryptic exon leads to a reduction in STMN2 expression. Thus, one strategy would be to restore normal STMN2 levels using a viral vector containing the correct DNA sequence, such as via adeno-associated viral (AAV) or lentiviral delivery. This proof of concept was shown when transduction of TDP-43 depleted iPSC-derived motor neurons with lentivirus carrying STMN2 restored STMN2 levels and also, importantly, restored axonal regeneration after axotomy [Stmn2 (Stmn2−/−) that the introduction of the human STMN2 gene was able to rescue motor deficits [SMN overexpression [UNC13A would be too large to be introduced via this methodology, as opposed to STMN2, which would theoretically be more amenable.The  axotomy . It was deficits . Howeverpression . Moreovepression . AnotherSOD1 and C9orf72 have been tested in ALS clinical trials . The former showed very encouraging results, but failed to reach significance in achieving primary clinical endpoints in a phase 3 trial, and the latter phase 1 trial showed no difference from placebo for clinical endpoints and in fact trended towards greater decline at the higher dose. These results highlight some of the caveats of ASO therapies [Other important gene-therapy strategies involve the prevention of erroneous RNA splicing. Compared to the above described gene delivery methods, these approaches aim to correct endogenous splicing and therefore avoid problems deriving from overexpression and toxicity. One method is via antisense oligonucleotide therapies (ASOs), which are short synthetic DNA sequences that are designed to selectively bind to pre-mRNA in cells. ASOs have come to the forefront as a new tool for numerous neurodegenerative diseases, including toxic gain of function gene-specific ALS subtypes, where they have been used to degrade mutant transcripts. ASOs designed to reduce protein produced from mutant herapies , 71. ASOherapies . One draRuminococcus flavefaciens), and using modified uridine-rich small nuclear RNA (snRNA) gene therapy, such as via U7 snRNA delivery, which has shown promise in DMD, and has entered its first clinical trial [Other effective splice switching strategies include systems that target pre-mRNA, but are stably expressed in the long-term, thereby preventing the need for repeated dosages, whilst also aiming to circumvent the above concerns regarding overexpression. These include CRISPR-Cas ribonuclease programming, such as via RfxCas13d (also named CasRx, from al trial –78. In aFinally, small molecule delivery has been developed as a further tool to alter splicing. A clear benefit of this therapeutic platform is the ability to deliver the treatment to patients orally. The first small molecule splicing modifier to be approved was Risdiplam for oral delivery in SMA. It was shown to increase SMN protein in the central nervous system and peripheral tissues in mice, and to restore functional SMN protein in patients with SMA –82.STMN2 and UNC13A that have been reported in humans are not present in mice. In fact, and interestingly, different UNC13A cryptic exons are present in mice [Homing in on correcting cryptic exon-related mis-splicing, a phase I clinical trial is planned to assess an ASO designed to restore STMN2 levels . Commonl in mice . Therefo in mice , 85. Ano in mice , 35. WhiWhilst blocking TDP-43 cryptic exons to restore gene expression appears to have a relatively clear mechanistic benefit, it remains to be seen how many and which cryptic exons need to be targeted for an effective therapy. Future experiments may seek to determine which cryptic exon inclusion events are pathologically relevant, impacting on a biological pathway that contributes to disease, as this may impact the development of therapies and potential ways to restore the expression of multiple mRNA targets, as opposed to one at a time.The past decade has seen leaps forward for people with gene-specific ALS-FTD, now with gene-based therapies coming into play. However, these would not benefit people with sporadic disease, making up the majority of patients with ALS-FTD, or those that have different pathogenic genes that are not amenable to treatment with available gene-based therapies. Given that almost all patients with ALS have TDP-43 pathology, the recent breakthroughs revealing that TDP-43 dependent mis-splicing of mRNA plays a major role in disease pathogenesis provides a fresh injection of hope into the field, not only for the ability to better understand molecular mechanisms, but also for the transformative potential with regard to biomarker development and therapeutics, desperately needed for patients living with this fatal disease. Important focuses going forward will be to establish which other cryptic exons, if any, are pathogenic players, and for the knowledge gained about cryptic exons to be transferred to and explored in other TDP-43 proteinopathies.


In this study, the integrated three-in-one  microsensor was made through the technology of the Micro-electro-mechanical Systems (MEMS) to measure three important physical quantities of the internal environment of the cold air pipe of the Heating, Ventilation and Air Conditioning (HVAC) in the factory, plan the installation positions of the integrated three-in-one microsensor and commercially available wind speed sensor required by the internal environment of the cold air pipe, and conduct the actual 310-h long term test and comparison. In the experiment, it was also observed that the self-made micro wind speed sensor had higher stability compared to the commercially available wind speed sensor (FS7.0.1L.195). The self-made micro wind speed sensor has a variation range of ±200 mm/s, while the commercially available wind speed sensor a variation range of ±1000 mm/s. The commercially available wind speed sensor (FS7.0.1L.195) can only measure the wind speed; however, the self-made integrated three-in-one microsensor can conduct real-time measurements of temperature and humidity according to the environment at that time, and use different calibration curves to know the wind speed. As a result, it is more accurate and less costly than commercially available wind speed sensors. The material cost of self-made integrated three-in-one microsensor includes chemicals, equipment usage fees, and wires. In the future, factories may install a large number of self-made integrated three-in-one microsensors in place of commercially available wind speed sensors. Through real-time wireless measurements, the self-made integrated three-in-one microsensors can achieve the control optimization of the HVAC cold air pipe’s internal environment to improve the quality of manufactured materials. Heating, Ventilation, and Air Conditioning (HVAC) is a system that uses mechanical equipment for ventilation. By introducing air from the outside and expelling air from the area, it could maintain the quality of indoor air and reduce moisture, odors, and pollutants in the air. Due to the need for introducing a significant amount of outdoor air, the internal environment of the HVAC cold air is affected, and wind speed, temperature, and humidity may have an internal effect. In addition, the geographical location and environment of the factory may also make a difference according to changes in temperature or short-term extreme climate (such as typhoons and heavy rain). Therefore, in order to improve the quality and reliability of manufactured materials of the factory, monitoring the internal environment of HVAC cold air ducts, such as wind speed, temperature, and humidity, and analyzing data using three essential physical quantities, are crucial research topics for finding optimal control parameters for the internal environment.The research aims to achieve real-time wireless monitoring of important information such as wind speed, temperature, and humidity within the HVAC cold air duct, and establish a complete database. It involves planning the various types of sensors required for the internal environment of the cold air duct, their installation location and quantities, evaluating and purchasing commercially available wireless sensors of different specifications, and developing a wireless flexible three-in-one  microsensor for software and hardware integration. The goal is to optimize the deployment of commercially available wireless sensors and self-made wireless flexible three-in-one microsensors, and collect important information to assist in field verification. This will achieve the optimization of the internal environment control of the HVAC cold air duct and improve the quality and reliability of process materials.3 to 25.99 gm3 as the temperature increased from 25 °C to 60 °C, with no significant change in water absorption. Beniwal et al. [Yi et al.  made a nl et al.  found thThe literature measures only a single physical quantity. We developed an integrated three-in-one microsensor that can measure the three important physical quantities of wind speed, temperature, and humidity at the same time. There is a relationship between the three physical quantities. We developed an integrated three-in-one  microsensor using MEMS technology and use Arduino for real-time monitoring. This will enable the deployment of self-made wireless three-in-one microsensors and the collection of important information to achieve the optimization of the internal environment control of HVAC air conditioning cold air ducts.At present, there are many kinds of wind speed sensors developed using MEMS technology. Generally, wind speed sensors can be divided into two categories: hot wire type wind speed sensors and calorimeter type wind speed sensors. Among them, hot wire wind speed sensors have a small size, high sensitivity, and high accuracy. Moreover, the design of its process, drive, and signal output circuit is relatively easy . Therefoρ) and temperature (T). in Equation (2), ρ is the resistivity at temperature T, ρ0 is the resistivity at reference temperature (T0), and α is the Temperature Coefficient of Resistance (TCR).The micro temperature sensor in this study belongs to Resistance Temperature Detector (RTD). It uses gold (Au) as the electrode material and conforms to the linear equation of Equation (2) between resistivity  could be obtained by substituting Equation (2) into Equation (3) of resistance law, where α here is the sensitivity of temperature (1/°C), which represents the change rate of resistance with each change of temperature per °C. It could be verified through this equation that the metal may expand and contract with the temperature change. In addition, the Steinhart-Hart Equation (SHHE) was derived by Cui et al. [The physical meaning of i et al. . It furt® PI 9305, FUJIFILM Electronic Materials Taiwan Co., Ltd., Hsin-Chu, Taiwan), which is strong in toughness, acid resistance, alkali resistance, corrosion resistance, and moisture absorption, as the moisture sensing film on the humidity sensor. Normally, such materials have non-conductive properties. When moisture absorbed by the moisture sensing film increases, the dielectric constant of the moisture sensing film may also increase with the rise in ambient humidity [The micro humidity sensor used in this study is a resistance humidity sensor. It uses a new negative photoresistance of polyimide  as the protective layer and dielectric layer. The protective layer is added to temperature and wind speed sensors to protect the circuits. An insulating layer is added for humidity sensors because the LTC 9305 is used as a humidity sensing material. If an insulating layer is added as a protective layer for humidity sensors, the humidity sensor will not be able to measure humidity. In terms of the manufacturing process, it mainly utilized deposition, lithography, and lift-off to superimpose multilayer films. Moreover, all micromechanical structures were made using thin film deposition, as shown in In this study, MEMS technology was used to integrate three sensing structures, namely temperature, humidity, and wind speed. In order to make the integrated three-in-one microsensor bendable, long-term non-damage and long-term measurement in the mechanism inside the HVAC cold air pipe, this study selected anti-corrosion, high temperature resistant, stretchable, and bendable Polyimide (PI) film as the substrate of the integrated three-in-one microsensor, with the thickness of 50 µm. It uses LTC(a)First, clean the PI film with organic solvents such as acetone and methanol, and then rinse it with deionized water to remove residual methanol, surface dust, and residual oil, in order to increase the adhesion of the thin film metal.(b)Coat with AZP4620 and use photolithography to define the electrode pattern of micro temperature, humidity, and wind speed sensors.(c)Then use an electron beam evaporator to deposit chromium and gold as adhesive layers and sensing electrode layers.(d)After lift-off using acetone, coat with LTC 9320 as a protective layer, and then use photolithography again to define the sensing area and terminals of micro temperature and wind speed sensors, which are covered and protected by the protective layer.(e)Coat with LTC 9305, and then use photolithography to define the sensing area of the micro humidity sensor, which is covered by a dielectric layer.The following are the order of the processes:The integrated three-in-one microsensor was cut to the smallest size to facilitate the placement of the internal fixing mechanism of the cold air pipe. Next, the integrated three-in-one microsensor was affixed to the ceramic substrate with conductive copper foil adhesive. Afterwards, the contact ends were strengthened and fixed with conductive silver adhesive, and the signal wires of the integrated three-in-one microsensor were welded to the ceramic substrate to connect the integrated three-in-one microsensor to the microprocessor on the computer side. The actual photo of the integrated three-in-one microsensor after signal wire welding is shown in The assembly of this microsensor had to conform to the fixed mechanism inside the factory’s HVAC cool air pipe and was expected to be able to resist the greasy environment inside the cold air pipe. Therefore, for the mechanism inside the cold air pipe, 3D printing was used to design the assembly as it could simultaneously fix the integrated three-in-one microsensor onto the mechanism. Finally, the integrated three-in-one microsensor was inserted into the 3D printed assembly, and the locking mechanism is shown in f is the voltage reading at the current wind speed, V0 is the voltage reading at 0 m/s, and ((Vf − V0)/V0) is the dimensionless variation of voltage reading.The wind-speed calibration range of the self-made micro wind speed sensor was 0 m/s to 10 m/s. The micro wind speed sensor might be affected by temperature. Therefore, the ambient temperature ranged from 5 °C to 40 °C during the calibration. Data were captured at wind speeds ranging from 0 m/s to 10 m/s every 5 °C and were measured repeatedly three times. In addition, through the dimensionless variation of voltage readings, the calibration diagram was obtained, as shown in f is the voltage reading at the current temperature, V0 is the voltage reading at 5 °C, and ((Vf − V0)/V0) is the dimensionless variation of voltage reading.The temperature calibration range of the self-made micro temperature sensor was from 5 °C to 40 °C, and the interval was 5 °C. Two self-made micro temperature sensors were put into the program controllable constant temperature and humidity machine. In addition, data were collected by Arduino every 5 °C and measured repeatedly three times. The relationship curve of temperature with the original voltage value read by Arduino was obtained, and the temperature calibration curve was completed. Then, the temperature calibration curve was illustrated using the dimensionless method, as shown in f is the voltage reading at the current humidity, V0 is the voltage reading at 70% RH, and ((Vf − V0)/V0) is the dimensionless variation of voltage reading.The humidity calibration range of the self-made micro humidity sensor was from 70% RH to 95% RH, and the interval was 5% RH. The micro humidity sensor was put into the program controllable constant temperature and humidity machine. Since temperature might affect the resistance value of the micro humidity sensor, the calibration was carried out in the temperature range of 5 °C to 40 °C, with an interval of 5 °C. In order to ensure the accuracy of the humidity and temperature displayed by the programmable constant temperature and humidity machine, this study used a commercially available multifunctional sensor that could measure humidity and temperature at the same time for calibration. Due to the hysteresis effect of moisture-sensing materials, there might be some differences in the resistance value under the same humidity in the process of humidification and dehumidification. Therefore, in this study, the micro humidity sensor was calibrated from low humidity to high humidity and from high humidity to low humidity. Furthermore, the dimensionless variation of voltage values was used to obtain the calibration diagram, as shown in The self-made integrated three-in-one microsensor and the commercially available wind speed sensor were actually installed inside the HVAC cold air pipe of the factory, as shown in ® LTC 9320) resistant to electrochemical corrosion and acid was used as the protective layer. This integrated three-in-one microsensor has the features of a small size, high sensitivity, grease resistance, and bendability. Additionally, it offers the benefits of measuring three critical physical parameters in real-time and simultaneously, namely wind speed, temperature, and humidity. It can be placed in any position, and it is cheap and has a fast response time. By placing the self-made integrated three-in-one microsensor inside the HVAC cold air pipe of the factory together with the commercially available wind speed sensor for field monitoring and data comparison, it could be known that the self-made integrated three-in-one microsensor had higher accuracy. Moreover, the reliability and durability of the self-made integrated three-in-one microsensor were further verified over 310 h in a long-time test. The commercially available wind speed sensor (FS7.0.1L.195) can only measure wind speed, while the self-made flexible three-in-one microsensor determines wind speed based on different calibration curves for environmental temperature and humidity, making it more accurate than the commercially available wind speed sensor (FS7.0.1L.195). In the future, in terms of wireless software design, additional IC chips need to be purchased to enable an Arduino board to connect to more sensors.In this study, MEMS technology was used to integrate the wind speed, temperature, and humidity microsensors onto the Polyimide (PI) substrate with a thickness of 50 µm, and the polyimide (Fujifilm Durimide


Depression is a mental disorder that significantly reduces quality of life, and the discovery of new drug targets is an urgent problem for modern neuroscience. Brain-derived neurotrophic factor (BDNF) and its receptors have been found to participate in mechanisms of depression and antidepressant drugs’ action. In this study, we focused on a less-studied truncated isoform of receptor TrkB: TrkB.T1. Initially, we noticed that the level of TrkB.T1 is low in the hippocampus of Antidepressant-Sensitive Cataleptics (ASC) mice, which are characterized by genetically determined depressive-like behavior in contrast to “normal” C57BL/6J mice. Next, overexpression of TrkB.T1 receptor in hippocampal neurons of ACS mice was induced to clarify the role of this receptor in mechanisms of depressive-like behavior. TrkB.T1 overexpression lowered BDNF protein concentration in the hippocampus. On the behavioral level, TrkB.T1 overexpression severely decreased aggression and enhanced social behavior. Additionally, this excess of receptor TrkB.T1 slightly promoted anxiety and depressive-like behavioral traits without affecting learning and memory. Our results show that this TrkB isoform participates in the control of aggression, anxiety, and depressive-like behavior and in the regulation of BDNF system functioning in ASC mice . Considering our findings, we believe that hippocampal receptor TrkB.T1 can be a drug target for the correction of behavioral pathologies. Ntrk2 transcript. These isoforms are the full-length tyrosine kinase receptor (TrkB.FL), the C-terminally truncated receptor (TrkB.T1), a C-terminally truncated Shc+ receptor (TrkB.shc), and a C-terminally truncated receptor retaining tyrosine kinase activity (TrkB-T-TK) [The Trk family of receptors includes TrkA, TrkB, and TrkC, which mediate the cellular effects of neurotrophins . TrkB iskB-T-TK) .NTR [Lacking the intracellular domain, TrkB.T1 cannot transduce signals to intracellular BDNF-initiated pathways. Nonetheless, TrkB.T1 has been found to have many functions. Having an extracellular domain identical to that of TrkB.FL, TrkB.T1 can bind BDNF with the same affinity and form homo/heterodimers with receptors TrkB.T1 and TrkB.FL . TrkB.T1NTR , which iNTR . ReceptoNTR . In the NTR . In gliaNTR .Since its discovery, studies on receptor TrkB.T1 have revealed its association with anxiety , aggress2A receptor. On the other hand, BDNF receptors have not been studied in this model yet.To discover the participation of receptor TrkB.T1 in depressive-like behavior, here we chose the Antidepressant-Sensitive Catalepsy (ASC) mouse strain: a genetic model of depression that has been developed by backcrossing predisposed-to-catalepsy CBA/Lac mice and catalepsy-prone AKR/J mice ,19. ASC Bdnf, Ntrk2, and Ngfr) and on mRNA levels of Mapk3, Creb1, and early response genes (Fos and Jun) required for long-term potentiation (LTP) induction in the hippocampus of ASC mice, which show genetically determined depressive-like behavior.In this work, we assessed the effects of TrkB.T1 overexpression in hippocampal neurons on different kinds of behavior using a wide range of behavioral tests  as well as on the expression of key genes of BDNF systems . All experimental animals were 2 months old. Behavioral tests were divided into two series, and the first one included the open field test, elevated plus maze, FST, and resident–intruder test. The second series consisted of the Morris water maze test and TST. In each test series, two groups of mice were evaluated: an experimental group (11 mice) and a control group (10 mice). An outline of the experiment is shown in In the resident–intruder test, 1.5-month-old mice of the CD1 strain (weight 25 ± 1 g) were used aside from ASC mice. Mature male mice of C57BL/6J strain  were used for comparative assessment of BDNF, TrkB, and TrkB.T1 mRNA levels. The animals were kept in the vivarium of the Institute of Cytology and Genetics SB RAS under standard laboratory conditions with a 12/12 h light/dark cycle and free access to feed and water.For fluorescence imaging, we employed three mice injected with adeno-associated virus (AAV) vector AAV-TrkB.T1-EGFP.With primers  containing recognition sites of restriction endonucleases BamHI and AgeI, a gene corresponding to TrkB.T1 was synthesized by means of murine cDNA as a template. The obtained sequence was digested with restriction endonucleases BamHI and AgeI  and ligated into the pAAV_Syn_EGFP vector to be expressed under the control of a synapsin (Syn) promoter . Sanger 2. The cells were split at 70% confluency, and the medium was refreshed every 2–3 days. HEK293FT cells were transfected with plasmids using polyethylenimine , following the manufacturer’s instructions.HEK293FT cells were subcultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% of fetal bovine serum , 1% of GlutaMAX , and 1% of a penicillin–streptomycin solution . The cells were incubated at 37 °C with 5% CO9 viral genomes per microliter).Packaging of plasmid AAV_Syn_TrkB.T1 or control plasmid pAAV_Syn_EGFP DNA into AAV capsids was performed by cotransfection of HEK-293FT cells with plasmids AAV-DJ  and pHelper  . Harvesthttp://labs.gaidi.ca/mouse-brain-atlas/?ml=-1.5&ap=-2&dv=2, accessed on 2 September 2021). Virus AAV_Syn_TrkB.T1 (AAV-TrkB.T1) or AAV_Syn_EGFP (control) was injected into the hippocampus bilaterally (1 μL per side) through a Hamilton syringe at a rate of 0.1 μL/min over a 10 min period. To minimize any drawback of the virus as the needle was removed, the needle was kept in place for further 2 min after the injection. After injections, the incision was closed with interrupted silk sutures, and the animal was placed in a warm cage.Animals were anesthetized with a solution (1 mL/kg i.p.) of 2,2,2-tribromoethanol  in 2-methyl-2-butanol  and placed in a stereotaxic frame . The skull was drilled bilaterally , thereby improving the contrast of an animal against the background. Mice were placed near the wall of the arena and allowed to freely explore the space for 5 min. Using EthoStudio software and hardware, the following parameters were automatically estimated: total distance traveled (m) and time spent at the center of the arena (%), the diameter of which was 27.5 cm. The numbers of rearings and groomings as well as average grooming duration (s) were determined manually. After each mouse, the arena was wiped with wet and dry wipes.Elevated plus maze test. The test setup consisted of four arms 30 cm long and 6 cm wide connected at a right angle and located at a height of 60 cm above the floor. One pair of arms had walls 20 cm high (closed arms), and the other pair had no walls (open arms). The arena was illuminated from above with diffused light. Mice were placed at the intersection of two arms, and movement was automatically recorded for 5 min using the EthoStudio software. The following parameters were automatically estimated: distance traveled (m), time spent in open and closed arms (%), and studied area of each arm (%). Peeks into closed arms and head dips were counted manually.The intermale aggression test. The test was performed within the resident–intruder paradigm as described previously [eviously ,26. A raTail suspension test. Mice were fixed with adhesive tape by the tail and hung on a horizontal bar at a height of 30 cm from a table surface. The number of immobilization episodes and latency time and duration of each episode were recorded using the EthoStudio software.Forced swim test. Mice were placed in a clear plastic box (15 × 25 cm) filled with water at 25 °C. After 2 min of adaptation, total immobility time and latency time of the first episode of immobility were recorded in the EthoStudio software Version 2.0 during 4 min [ng 4 min .Morris water maze test. The setup comprised a round pool 110 cm in diameter with walls 30 cm high. The pool was filled with water , and powdered milk was added to make it opaque. We tracked each animal by transmitted (inverted)-lighting techniques developed for the open field test. In the EthoStudio software, the pool was divided into four sectors. A glass round escape platform (5 cm diameter and 14.5 cm height) was located 1 cm below the water surface near the center of the lower right quadrant of the maze. A black rectangle measuring 6 × 12 cm  was glued to the pool wall closest to the platform. Mice were automatically tracked by means of the EthoStudio software.Each training period lasted 4 days, and each session contained three trials. During the training, mice were allowed to search for the platform for 60 s. At the end of each test, mice were placed on the platform and held (if necessary) there for 15 s. Measured parameters were latency to find the platform (s), distance traveled (m), and total distance to the platform (m), which was calculated as the sum of distances from the mouse to the platform at each time point. The average value of three tests was subjected to further analyses.On the fifth day, the platform was removed, mice were placed at the center of the pool, and their movements were recorded for 60 s. Time spent in the target sector (%) was estimated, and then the average value for the three trials was calculated. A statistically significant excess of time spent in the target sector over a random level (25%) indicated that the mouse remembered the location of the platform.Two days after the behavioral testing, the animals were decapitated, and hippocampi were isolated on ice, frozen in liquid nitrogen, and stored at −80 °C until subsequent procedures.2, 2 µL of mixture of forward and reverse primers, and 0.16 µL of Taq DNA polymerase, and adjusted the volume to 20 µL with deionized H2O. Two types of standard were used for quantitative analysis of mRNA levels: external and internal. An internal standard (mRNA of the rPol2 gene) was employed to make sure that the reverse transcription worked and as a basis for calculating mRNA level of the studied genes. In preliminary experiments, no differences were found in the level of rPol2 mRNA among the control and experimental groups. A series of dilutions of DNA (of a known concentration) isolated from the mouse liver served as an external standard, which made it possible to check whether the PCR worked and to determine the number of mRNA copies of the studied genes in the samples. Gene expression is presented as the ratio of cDNA of an analyzed gene to 100 copies of rPol2 cDNA [We mixed a 1 µL aliquot of cDNA with 2 µL of PCR buffer (containing SYBR green I), 2 µL of 25 mM dNTP, 2 µL of 25 mM MgClol2 cDNA . Meltingg for 15 min at 4 °C, the supernatant was collected and centrifuged at 20,000× g at 4 °C for an hour. The cytosolic fraction was collected, and the precipitate remaining in the tube was resuspended in homogenizing buffer (see above). Both fractions were stored at −80 °C. Protein concentration was assessed spectrophotometrically using the Pierce BCA Protein Assay Kit  and a NanoDrop 2000C spectrophotometer . The samples were adjusted to equal concentrations (1 mg/mL) using 2× Laemmli’s buffer and heat denatured for 10 min at 95 °C. Extracts  separated by SDS-PAGE were blotted onto a nitrocellulose membrane  with the help of a Trans-Blot Turbo Transfer System . The membranes were blocked in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST) containing 5% of nonfat dry milk (NFDM-TBST) for 1 h, washed, and then incubated with primary antibodies shown in Membrane proteins were isolated by homogenizing samples in a buffer consisting of 300 mM sucrose, 10 mM Tris-HCl pH 7.2, 1 mM EDTA, 5 mM β-mercaptoethanol, and protease inhibitors . Then, the samples were centrifuged at 500× In the fifth week after injection, mice were transcardially perfused with phosphate-buffered saline (PBS) and a 4% paraformaldehyde solution. Brains were excised, postfixed with 4% paraformaldehyde for 6 h, and immersed in 20% sucrose in PBS for 2 days. Using a cryostat , 14 µm slices were obtained, and finally mounted in an antiquenching medium with DAPI  followed by examination under a Zeiss AxioImager2 microscope with 10× and 40× air-immersion objectives. The mice used for the imaging were not subjected to the behavioral testing.t test if the data were normally distributed. Nonparametric Mann–Whitney U test was utilized when data were non-normally distributed. The results of the Morris water maze test were subjected to repeated measures analysis of variance (ANOVA) with Fisher’s test post hoc comparison. Analysis of covariance (ANCOVA) was applied to determine whether immobility in the tail suspension test and social activity in the resident–intruder test depended on reduced locomotor activity in the open field test.All data were tested for normality of distribution by Shapiro–Wilk, Kolmogorov–Smirnov, D’Agostino and Pearson, and Anderson–Darling tests. Gene expression data and the results of open field, elevated plus maze, tail suspension, forced swim, and resident–intruder tests were processed by Student’s p = 0.004) was reduced in the hippocampus of ASC mice, in agreement with reports on reduced BDNF expression in patients with major depressive disorder [p = 0.81), the TrkB.T1 mRNA level proved to be downregulated (t(10) = 3.08, p = 0.01) in the hippocampus of ASC mice compared to C57BL/6J mice  in the hippocampus of “depressive” (ASC) and “nondepressive” (C57BL/6J) mice. We revealed that the BDNF mRNA level (t(12) = 3.54, disorder . Of note/6J mice A. As farp < 0.0001) and protein levels (t(15) = 3.37, p = 0.004; To identify the role of hippocampal TrkB.T1 in mechanisms underlying depressive-like behavior of ASC mice, we induced TrkB.T1 overexpression by injecting the hippocampi with a recombinant AAV carrying the gene expressing TrkB.T1. Five weeks after the injection, we performed behavioral testing, and next confirmed TrkB.T1 overexpression in the hippocampus both at mRNA (t(18) = 5.22, p = 0.02) (p = 0.75); the same was true for the time spent in open arms (t(18) = 1.25, p = 0.40).Mice with TrkB.T1 overexpression spent significantly less time at the center of the arena in the open field test (t(19) = 2.48,  = 0.02) . This fip = 0.003). On the other hand, the elevated plus maze did not reveal any differences in exploratory activity, assessed by means of peeks (t(18)= 0.83, p = 0.41), latency to the first peek  and the number of head dips   = 3.46,  = 0.98) .p = 0.05). This result was confirmed by the elevated plus maze test (t(18) = 2.26, p = 0.036).Mice with TrkB.T1 overexpression also showed a strong tendency toward shorter distance traveled in the open field test (t(19) = 2.00, p = 0.28), explored area in closed arms of the elevated plus maze , explored area in open arms , and grooming parameters , duration , and latency time of the first grooming episode (t(19) = 0.16, p = 0.16)).Other parameters analyzed in both tests were not affected by TrkB.T1 overexpression: explored area in the open field test  in the tail suspension test (p = 0.59) and the number of immobility episodes (t(21) = 0.51, p = 0.61) did not differ between the two groups. To investigate whether immobility in the tail suspension test depends on diminished locomotor activity, ANCOVA was performed with total traveled distance in the open field test being a continuous predictor of immobility in the tail suspension test. In this case, AAV-TrkB.T1-EGFP–injected mice still demonstrated a significantly higher level of immobility in the tail suspension test as compared to corresponding control animals .Mice with TrkB.T1 overexpression showed longer immobility time as compared to the control (t(21) = 2.61, ion test . Neverthp = 0.70), latency to the first immobility episode (t(16) = 1.73, p = 0.10), and the number of immobility episodes (t(16) = 0.34, p = 0.10)), we assumed that the TrkB.T1-induced prodepressive effect is too weak to be detected by both tests.Because the influence of receptor TrkB.T1 on immobility was not confirmed in the forced swim test (immobility duration (t(14) = 0.54, p = 0.0004; p = 0.0003; p < 0.0001; p = 0.004). The number of social contacts (t(18) = 0.23, p = 0.63) was not significantly affected by TrkB.T1 overexpression. At the same time, latency to the first social contact (t(18) = 2.00, p = 0.06) manifested a strong decreasing trend in the experimental group. In ANCOVA with traveled distance as a continuous predictor, we also detected a significant impact of TrkB.T1 overexpression on the number , duration , and latency time  of aggressive contacts and on the duration of social interactions .The resident–intruder test revealed that TrkB.T1 overexpression significantly diminishes the number of aggressive contacts , distance traveled by a mouse (p < 0.01 and p < 0.05 for groups AAV-EGFP and AAV-TrkB.T1), and cumulative distance between the mouse and the platform at each time point (p < 0.05 and p < 0.01 for groups AAV-EGFP and AAV-TrkB.T1)), indicating that both groups successfully learned the task. For each examined parameter, we found a significant effect of the training day on the distance traveled , cumulative distance , and latency to find the platform .We carried out the Morris water maze test to evaluate the effects of receptor TrkB.T1 on learning and memory . All parp ˃ 0.05), cumulative distance , and latency to find the platform ). This meant that the two groups had a similar ability to learn, and the same was true for the ability to memorize.By contrast, the influence of TrkB.T1 overexpression was not significant  and AAV-TrkB.T1 (p < 0.01)) and the influence of training was confirmed . Therefore, the effect of TrkB.T1 overexpression on spatial memory and learning was not detectable .On retention day (the test without the platform), both groups spent significantly more time in the target sector compared to the other sectors (AAV-EGFP (p = 0.02), while transcription of its gene remained unchanged (t(16) = 1.56, p = 0.13; Ntrk2 gene (mRNA (t(15) = 0.48, p = 0.63) and protein (t(17) = 1.29, p = 0.21)) was not affected by TrkB.T1 overexpression. Neither was the expression of the receptor of proBDNF, i.e., receptor p75NTR encoded by the Ngfr gene , protein (t(17) = 0.64, p = 0.52); TrkB.T1 overexpression significantly lowered the BDNF protein level in mice of the experimental group (t(17) = 2.39, p = 0.26), Mapk3 , and early response genes: Fos (t(17) = 0.61, p = 0.54) and Jun (t(17) = 0.67, p = 0.50; We did not find any impact of TrkB.T1 overexpression on mRNA levels of LTP-related genes: Creb1 (t(18) = 1.15, Our preliminary study suggests that transcription of genes of TrkB.T1 and BDNF is significantly lower in the hippocampus of ASC mice  than in “nondepressive” C57BL/6J mice. Intrigued by this novel finding, we induced hippocampal TrkB.T1 overexpression in ASC mice using an AAV-TrkB.T1 construct. Then, we analyzed behavioral patterns and assessed the expression of genes that may be responsible for changes in behavior. Overexpression of TrkB.T1 did not cause regulatory compensation in terms of receptor TrkB.FL expression in the hippocampus of the studied mice, implying that the observed behavioral and genetic alterations are TrkB.T1-induced.Open field test results showed that overexpression of TrkB.T1 receptor has a mild effect on anxiety, judging by less time spent at the center of the arena and more time spent near the wall of the arena by mice from experimental group (TrkB.T1 overexpression). To rule out a possible false positive result, we measured the explored area of the open field in both groups. Because there were no differences in this parameter between the groups, it is safe to assume that the difference in time spent at the center is actually due to a difference in anxiety rather than changes in locomotor or exploratory activity. This result, however, failed to be replicated in the elevated plus maze test: the two groups spent similar periods in closed arms, and the same was true for open arms. Head dips, which are an indicator of anxiety , also diWe noticed the decrease in the number of rearings in TrkB.T1 overexpressed mice. Given that the explored area of arena was not changed, the decrease in the number of rearings in the open field test may also be due to greater anxiety . In suppOf note, TrkB.T1 knockout mice tend to spend less time in the center of the arena in the open field test and spend significantly less time in the open arms of the elevated plus maze . Thus, TIn addition, the overexpression of TrkB.T1 reduced the distance traveled by the experimental group in the elevated plus maze. Mice with TrkB.T1 overexpression also tended to have a shorter distance traveled in the open field test. On the other hand, the locomotor activity measured by the Morris water maze test did not differ between the groups. It is noteworthy that the knockout of receptor TrkB.T1 in C57BL/6J mice does not affect their locomotor activity . TrkB.T1FST and TST are designed to assess depressive-like behavior in rodents. Our results uncovered an increase in immobility time in the TST but not in the FST during hippocampal TrkB.T1 overexpression. The discrepancy between these two closely related tests is explained by the fact that both tests, although similar in the constructs they are designed to assess, are probably different in terms of the biological substrates underlying the observed behavior . Taking Mapk3, Creb1, and early response genes (Fos and Jun) required for LTP induction [The Morris water maze test revealed that hippocampal receptor TrkB.T1 does not affect learning dynamics and spatial memory in ASC mice. These results are consistent with unchanged mRNA levels of the nduction ,39. Our nduction ,12. On tnduction . In the nduction .We found that the baseline level of BDNF in ASC mice is lower than that in “nondepressive” C57BL/6J mice. This is expected because a depressive phenotype is known to be associated with BDNF deficiency . For exaAdditionally, we noticed a dramatic effect of receptor TrkB.T1 on aggression and social behavior. Overexpression of TrkB.T1 (i) reduced the number and duration of aggressive contacts, (ii) increased latency time of the first aggressive contact, (iii) extended the duration of social contacts, and (iv) diminished latency time of the first social contact. The antiaggressive effect of TrkB.T1 overexpression along with the decline of BDNF levels is consistent with the findings of Ilchibaeva and coauthors , who havWe showed that receptor TrkB.T1 is involved in mechanisms underlying anxiety, depressive-like behavioral traits, and aggression in ASC mice, which are genetically predisposed to depressive-like behavior. We confirmed for the first time that receptor TrkB.T1 regulates the BDNF level in neuronal cells. This knowledge means that TrkB.T1 should be taken into consideration in further studies on behavioral pathologies and underlying mechanisms.


Using the hybrid electrodes, thin-film transistors (TFTs) were fabricated, which exhibited an electron mobility of ~6.7 cm2 V−1 s−1, a current on-off ratio of ~1.04 × 107, and a subthreshold voltage of ~0.122 V/decade. These electrical properties are comparable with those of TFTs fabricated using Al electrodes. This suggests the possibility of customizing flexible transparent electrodes within a carbon nanomaterial system.In this study, we prepared flexible and transparent hybrid electrodes based on an aqueous solution of non-oxidized graphene and single-walled carbon nanotubes. We used a simple halogen intercalation method to obtain high-quality graphene flakes without a redox process and prepared hybrid films using aqueous solutions of graphene, single-walled carbon nanotubes, and sodium dodecyl sulfate surfactant. The hybrid films showed excellent electrode properties, such as an optical transmittance of ≥90%, a sheet resistance of ~3.5 kΩ/sq., a flexibility of up to  Flexible transparent electrodes have attracted significant interest in the field of wearable devices, such as electronic skins, foldable photovoltaics, soft robots, and flexible displays ,19,20,21With excellent optical, electrical, and mechanical properties, one- and two-dimensional carbon nanomaterial composites are finding applications as flexible transparent electrodes. In particular, a very small amount of such materials is needed to fabricate conducting electrodes, as high aspect ratios of one-dimensional CNTs lead to low percolation thresholds. However, CNT network films have large contact resistance from the percolation of charges through their junctions ,5,6. In Generally, graphene solutions are prepared by dispersing graphene oxide (GO) using Hummers’ method and directly exfoliating graphene flakes in organic or inorganic solvents ,23,24,25In this study, we developed an aqueous solution process for obtaining flexible transparent electrodes based on carbon nanomaterials. Using this method, we synthesized a high-quality, non-oxidized aqueous graphene solution from highly ordered pyrolytic graphite (HOPG) via the halogen intercalation method. We also prepared a hybrid aqueous solution of non-oxidized graphene and SWNT, which exhibited good dispersity and long-term stability. We then used this solution to fabricate hybrid film electrodes. We evaluated the mechanical, electrical, and optical properties of the hybrid electrodes with various graphene to SWNT ratios and film thicknesses. Furthermore, we fabricated fully transparent indium gallium zinc oxide (IGZO) thin-film transistors (TFTs) using the hybrid electrodes.3(CH2)11OSO3Na)  were purchased from Sigma Aldrich .Highly ordered pyrolytic graphite (HOPG) (Grade SPI-2 #466HP) was purchased from SPI . Raw HiPco single-walled carbon nanotubes (R grade SWNT) were purchased from Unidym . Natural graphite (325 mesh) was purchased from Alfa Aesar . Iodine monochloride (ICl) (reagent grade 98%) and sodium dodecyl sulfate (SDS)  and ICl  of 10 mg were introduced into a glass reactor and sealed with a Teflon cap. The reactor was then immersed in a silicon oil bath at 160 °C for 54 h. After the reaction between the graphite and intercalant, the as-prepared graphite intercalation compound (GIC) was immediately placed in a quartz tube at 800 °C with 60 sccm of Ar flow for 10 min to obtain expanded graphite. Subsequently, the furnace was cooled to room temperature while argon continued to flow. Expanded graphite of 10 mg was added in 1 wt.% aqueous SDS solution of 10 mL. To disperse and break the expanded graphite, the solution was homogenized for 1 h and then bath-sonicated for 30 min, followed by slow-speed centrifugation at 2100 rpm for 20 min. Finally, the top half of the dispersion was extracted with a vacuum pipette (~8 mL) and retained for use.HOPG of 20 mg  and Raman spectroscopy  with a laser excitation wavelength of 532 nm. The thickness and optical transmittance of the graphene/SWNT electrodes were examined using a surface profiler  and a UV-vis-NIR spectrometer . The surface morphology of the graphene/SWNT electrodes was visualized using field-emission scanning electron microscopy . X-ray photoelectron spectroscopy  measurements were carried out using a monochromatic Al Kα X-ray source (1486.6 eV) to determine the chemical compositions of the graphene. The work function was measured using a Kelvin probe system . The sheet resistance was measured using a four-point probe system .2O3 (100 nm) was deposited for the top gate dielectric via atomic layer deposition (ALD) at 130 °C under 10−2 Torr followed by thermal treatment at 300 °C for 30 min under ~10−6 Torr. A transparent active layer was synthesized with spin-coating of IGZO (20 nm), followed by thermal treatment at 150 °C for 5 min and at 400 °C for 2 h. Source and drain electrodes were deposited using the graphene/SWNT solution by spray-coating followed by curing at 80 °C for 5 min. The channel regions 2 mm in width and 200 μm in length were patterned with shadow mask. Subsequently, the TFTs were soaked in ethanol at 50 °C for 5 min and then dried with N2 blowing, followed by heating in an oven at 50 °C for 5 min. For the comparison of the electrical properties of the TFTs, IGZO TFTs were fabricated using Al electrodes. The electrical properties of these devices were characterized using a semiconductor parameter analyzer  under ambient conditions.Fully transparent TFTs were fabricated on a glass substrate. For the gate electrode, ITO (150 nm) was deposited on glass by sputtering at room temperature. Subsequently, a layer of Al−1), D band (~1330 cm−1), G− band (~1540 cm−1) (or Breit–Wigner–Fano), G band (~1590 cm−1), and 2D band (~2640 cm−1) of SWNT ..−1), D  is summarized in ε = 3.6% (bending radius (R) = 1.4 mm). The sheet resistances of the SWNT- and the non-oxidized graphene/SWNT-based flexible electrodes fluctuated slightly within ±1% due to the bending strain. However, the hybrid electrodes exhibited better mechanical stability against repeated bending cycles (ε = 2.0% (R = 2.5 mm)) than the SWNT-based electrodes , current on-off ratio (Ion/Ioff), and subthreshold swing (SS) of the devices were estimated to be ~6.7 cm2 V−1 s−1, ~1.04 × 107, and ~0.122 V/decade, respectively  and high mechanical stability, as indicated by a sheet resistance of less than 1% even after 103 bending cycles at ε = 2.0% (R = 2.5 mm). Furthermore, the IGZO thin-film transistors fabricated using the hybrid electrodes exhibited device characteristics comparable with those of the TFTs fabricated using Al electrodes, with an electron mobility of ~6.7 cm2 V−1 s−1, a current on–off ratio of ~1.04 × 107, and a subthreshold voltage of ~0.122 V/decade. This demonstrates a significant step toward the application of carbon nanomaterials in flexible transparent device components for next-generation wearable devices.In this study, we prepared and investigated the properties of flexible transparent electrodes obtained using non-oxidized graphene/SWNT aqueous solutions. Owing to minimal defects in the non-oxidized graphene, 60 nm-thick hybrid film containing 30 wt.% graphene showed an optical transmittance of more than 90% and sheet resistance of ~3.5 kΩ/sq. In addition, the film exhibited excellent flexibility, with no electrical change up to


The quadratus lumborum (QL) block is an ultrasound-guided regional anesthesia technique that aims to provide analgesia to the abdomen. Previous studies show that this technique may fail to provide analgesia in the cranial abdominal wall in dogs. The QL block is a difficult technique with the risk of damaging vital structures. The objective of this study is to assess a new latero-ventral approach to the QL (LVQL), which could be easier and safer to perform than the interfascial approach (IQL) widely reported. Another objective is to evaluate the distribution of high-volume injections (0.6 mL/kg) of dye/contrast achieved by both approaches. Six thawed dog cadavers were used to randomly perform the LVQL in one hemiabdomen and the IQL in the other. Distribution of the injectate throughout the ventral branches of the spinal nerves from T10 to L4 and the sympathetic trunk (CT and dissection) was assessed. The two approaches consistently stained L1-L3 ventral branches, which is compatible with obtaining somatic analgesia in the middle and caudal abdominal wall. The administration of high-volume injections did not result in a more cranial distribution of the injectate compared to previous studies. The sympathetic trunk was more consistently stained by the IQL, therefore, the LVQL may not promote visceral desensitization of the abdomen.The QL block is a high-level locoregional anesthesia technique, which aims to provide analgesia to the abdomen. Several approaches of the QL block have been studied to find out which one allows a greater distribution of the injectate. The aim of this study is to compare the traditional interfascial QL block (IQL) with a new latero-ventral approach (LVQL). We hypothesize that this new approach could be safer and easier to perform, since the injectate is administered more superficially and further away from vital structures. Our second objective is to assess whether a higher volume of injectate (0.6 mL/kg) could reach the ventral branches of the last thoracic nerves, leading to a blockade of the cranial abdomen. Six thawed canine cadavers (12 hemiabdomens) were used for this purpose. Both approaches were performed in all cadavers. A combination of methylene blue/iopromide was administered to each hemiabdomen, randomly assigned to the LVQL or IQL. No differences were found regarding the ease of perform the LVQL with respect to IQL. The results show that both techniques reached the ventral branches from L1 to L3, although only the IQL consistently stained the sympathetic trunk (5/6 IQL vs. 1/6 LVQL). The use of a higher volume did not enhance a more cranial distribution of the injectate. The quadratus lumborum (QL) block is an ultrasound-guided regional anesthesia technique that aims to block the ventral branches of the thoracolumbar nerves and sympathetic trunk . SeveralThe QL block was first described by Blanco et al.  as a varQuadratus lumborum block is a complex technique with a significant risk of damaging abdominal structures ,18. The This study intends to assess a new approach to the QL block, in which the injectate is administered lateral and ventral to the quadratus lumborum muscle (LVQL), closer to the skin surface and further away from important abdominal structures, to increase the ease and safety of the technique. A second objective is to assess the distribution of a high volume of injectate (0.6 mL/kg), comparing the LVQL to the interfascial QL block (IQL) as described by Garbin et al. . We hypoThe Biosafety Committee in Experimentation (CBE 433/2021) and the Ethical Committee for Animal Experimentation (CEEA 740/2021) of the University of Murcia approved this essay. Six canine cadavers (12 hemiabdomens) were used in this research. Cadavers with skin or muscle lesion in the lateral aspect of the thoracolumbar area, or vertebral alterations were excluded from the study. The animals were humanely euthanized for reasons unrelated to the study and immediately frozen. The cadavers were thawed at room temperature 48 to 72 h before the procedures.−1 of a solution made up of the same amount of methylene blue  and radiopaque contrast medium iopromide  was injected in each hemiabdomen. The same researcher (Eliseo Belda) performed all the injections.Two different approaches to the QL block (LVQL and IQL) were compared. Both approaches were performed in all the dogs. The technique to be performed in each hemiabdomen was randomly assigned, which was determined by extracting a code from a sealed envelope. To perform the study, cadavers were placed in lateral recumbency and an extensive clipping of the thoracolumbar area was carried out. The injections were made under ultrasound guidance, using a linear array  and echogenic needles . A volume of 0.6 mL kgTo perform both approaches, the ultrasound probe was placed transversal to the longitudinal axis of the spine, caudal and parallel to the last rib, at the level of L1. The array was slipped dorsally and tilted down until visualization of the target structures: erector spinae muscles (ESP), external oblique muscle (EO), internal oblique muscle (IO), transverse abdominal muscle (TA), transverse process of L1 (TP), transverse abdominal muscle aponeurosis (TAp), QL muscle, psoas minor muscle (PM) and vertebral body of (VB) L1, could be achieved .Once those structures were identified, the needle was inserted in plane in a ventro-dorsal, latero-medial direction. After passing over the EO and IO, the tip of the needle was directed towards the TAP which offered slightly more resistance. The perforation of the TAP generates a “pop” sensation. Sonographic images and videos were recorded, and their quality was subjectively analyzed later  as good , moderate (QL and PM margins were not clearly identified) and poor . The visualization of the needle tip at the moment of the injection was also registered.The targeted injection site in this approach was the latero-ventral aspect of the QL, on its border with the TAP. To perform the LVQL, the needle was stopped immediately after piercing the TAP, and the dye/contrast solution injected .The injection site of this approach was the interfascial plane situated between the QL and PM . This plFollowing the administration of the injectate, cadavers were taken to a computed tomography (CT) scan  to evaluate contrast distribution. To perform the CT, cadavers were placed in sternal recumbence and 3 mm thickness slices were obtained at vertebral levels from T8 to L7. Images were subsequently reconstructed and analyzed by two radiologists .Finally, the cadavers were subjected to anatomical dissection , the technique is explained in detail elsewhere . Brieflyp < 0.05.The statistical tests were performed using SPSS, version 24.0 . A Shapiro–Wilk test was used to assess the normality of the distribution. The distribution of dye/contrast in the CT and anatomical dissection studies between the two treatments were evaluated by a Yuen’s test. The quality of the ultrasound images and visualization of the needle tip were analyzed by a Fisher’s test. Mean ± standard deviation, median (range) or numbers of animals were chosen to express the data as deemed most appropriate in each case. Data are expressed as mean ± standard deviation, median and range or number of animals, as it was considered more appropriate in each case. Differences were considered statistically significant when Cadavers weighed 27.7 ± 12.5 kg and presented a median body condition score (BCS) of 4 (3–5) out of 9.p = 0.5). The target structures were found in all cases.The LVQL technique resulted in images classified as moderate (1/6) or good (5/6) accordingly to their quality. The IQL approach produced images of moderate (3/6) or good (3/6) quality. There were no statistical differences in image quality between the studied approaches (p = 0.85). In the case where the needle tip could not be observed, the correct positioning of the needle was confirmed after feeling two “pop” sensations as described before, as well as for the expected distribution of the injectate between QL and PM during the injection. The tip of the needle was observed at the time of injection in 6/6 cases for LVQL and in 5/6 for IQL. No statistically significant differences were found between the two approaches (p = 0.74). In the LVQL, the contrast media was mainly distributed within the ventral and lateral aspects of the QL. Regarding the IQL, the contrast media was observed surrounding the QL and PM muscles, as well as the sympathetic trunk area  VB, from T13 to L7, and in the IQL, 7.5 (5–9) VB, from T12 to L7 . No statunk area . One hemp = 0,03). The ventral branches of the spinal nerves in the LVQL were stained in a more distal portion, while the IQL dyed the nerves closer to their roots (p = 0.0011)  .The results of this study do not support our initial hypothesis, as both approaches were feasible and with a similar degree of difficulty. The studied approaches (LVQL and IQL) could promote the blockade of L1–L3 ventral branches. However, despite using high volume 0.6 mL/kg) injections, neither technique was able to consistently stain the ventral branches of the last thoracic spinal nerves (T10–T13), which are responsible for the innervation of the cranial abdominal wall . Therefo mL/kg inIt was also hypothesized that because the site of injection selected for the LVQL approach was located closer to the skin surface, this approach could be easier to perform than the IQL. However, no differences were found comparing the quality of the images and the visualization of the needle tip of both techniques. The IQL could be more difficult to perform in larger and obese dogs, due to the increased distance from the probe to the injection site. Most of the cadavers included in our study presented a low to moderate BCS (4 out of 9). This fact, together with the wide clinical expertise of the researcher in charge of performing the ultrasound-guided administration of the injectate, could have biased these results. It must also be considered that, in one out of six hemiabdomens in the IQL approach, the tip of the needle could not be visualized. With this being a cadaveric study, the position of the tip of the needle was inferred from the feeling of the two “pop” sensations. This practice cannot be recommended in live animals, as it is mandatory to visualize the tip of the needle during the administration of the drugs to avoid vascular, neural or intracavitary injections. Other safety measures that should be undertaken before the administration of the drugs are an aspiration and a resistance to injection test, to avoid intravascular or intraneural injections, respectively ,21.The site of injection for the LVQL approach was selected bearing in mind its proximity with the trajectory taken by the ventral branches of the first lumbar spinal nerves. These nerves cross through the interfascial space formed by the QL and PM muscles to run along the ventral and ventro-lateral aspects of the QL, piercing the TAP . Garbin The CT study revealed a wide distribution of injectate. In several cadavers of both groups, a caudal distribution of iopromide that reached L7 was observed. These findings are similar to previous descriptions ,6. In onThe use of a larger volume of injectate than that employed by Garbin et al.  did not In our study, the LVQL approach did not consistently stain the sympathetic trunk, while the IQL presented a more constant spread from L1 to L3 levels. These results show that the LVQL approach may provide a similar analgesic effect to those reported for a TAP block ,10,12. TIn 50% of cases in the IQL group, the genitofemoral nerve was stained. This nerve is responsible for the sensitive innervation of the proximal aspect of the medial thigh, skin of pudendal region, prepuce, mammary gland, vaginal tunic and vaginal process ,27. A pr−1 per hemiabdomen was chosen. This high volume must be considered when local anesthetics are administered, in order to avoid its systemic toxicity [Our study has several limitations. The results could be biased by the small number of cadavers available. The injections were carried out mostly in lean cadavers. Probably, the quality of the images obtained from larger or obese animals would be different because the ultrasonographic appearance of important structures to guide the block could be impaired in these types of patients. As the QL block is an operator-dependent technique, the fact that the ultrasound studies were performed by a researcher with high-clinical experience could also bias the results, particularly those comparing the ease of both approaches ,3,4,5. Ttoxicity ,29,30,31In conclusion, the LVQL would be a suitable option to provide analgesia to the abdominal wall, but not to the abdominal viscera. Despite the high volume of injectate administered in this study, the injections did not consistently reach the ventral branches of the last thoracic nerves in any group. Therefore, none of these approaches may be recommended to provide analgesia for the cranial abdominal wall. More clinical trials are necessary to test the QL block in live animals, to determine its analgesic effects and the extension of the blockade at both somatic and visceral levels.