Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability-Brief Report.

BACKGROUND: Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. METHODS: To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. RESULTS: AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. CONCLUSIONS: Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Reduced cardiac antioxidant defenses mediate increased susceptibility to workload-induced myocardial injury in males with genetic cardiomyopathy.

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking ß-sarcoglycan (ß-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this ß-SG-/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in ß-SG-/- mice. Male ß-SG-/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female ß-SG-/- hearts. This protection of females was independent of ovarian hormones. Male ß-SG-/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of ß-SG-/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male ß-SG-/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male ß-SG-/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.

Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.

RATIONALE: The initial hypertrophy response to cardiac pressure overload is considered compensatory, but with sustained stress, it eventually leads to heart failure. Recently, a role for recruited macrophages in determining the transition from compensated to decompensated hypertrophy has been established. However, whether cardiac resident immune cells influence the early phase of hypertrophy development has not been established. OBJECTIVE: To assess the role of cardiac immune cells in the early hypertrophy response to cardiac pressure overload induced by transverse aortic constriction (TAC). METHODS AND RESULTS: We performed cytometry by time-of-flight to determine the identity and abundance of immune cells in the heart at 1 and 4 weeks after TAC. We observed a substantial increase in cardiac macrophages 1 week after TAC. We then conducted Cite-Seq single-cell RNA sequencing of cardiac immune cells isolated from 4 sham and 6 TAC hearts. We identified 12 clusters of monocytes and macrophages, categorized as either resident or recruited macrophages, that showed remarkable changes in their abundance between sham and TAC conditions. To determine the role of cardiac resident macrophages early in the response to a hypertrophic stimulus, we used a blocking antibody against macrophage colony-stimulating factor 1 receptor (CD115). As blocking CD115 initially depletes all macrophages, we allowed the replenishment of recruited macrophages by monocytes before performing TAC. This preferential depletion of resident macrophages resulted in enhanced fibrosis and a blunted angiogenesis response to TAC. Macrophage depletion in CCR2 (C-C chemokine receptor type 2) knockout mice showed that aggravated fibrosis was primarily caused by the recruitment of monocyte-derived macrophages. Finally, 6 weeks after TAC these early events lead to depressed cardiac function and enhanced fibrosis, despite complete restoration of cardiac immune cells. CONCLUSIONS: Cardiac resident macrophages are a heterogeneous population of immune cells with key roles in stimulating angiogenesis and inhibiting fibrosis in response to cardiac pressure overload.

Neuroimaging findings in paediatric patients with sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive haemoglobinopathy, which manifests as multisystem ischaemia and infarction, as well as haemolytic anaemia. The morphological changes of red blood cells (RBCs) that promote ischaemia/infarction as the main multi-systemic manifestation, with associated vasculopathy, may also lead to haemorrhage and fat embolisation. Bone infarctions, whether of the skull or spine, are relatively common with subsequent increased infectious susceptibility. We present a broad spectrum of brain and spine imaging findings of SCD from a level III paediatric hospital in Lisbon, between 2010 and 2022. Our aim is to highlight brain and spine imaging findings from a serial review of multiple patients with SCD and respective neuroimaging characterisation.

The Emerging Role of B Cells in the Pathogenesis of NAFLD.

NAFLD is one of the leading causes of abnormal liver function worldwide. NAFLD refers to a group of liver conditions ranging from nonalcoholic fatty liver to NASH, which involves inflammation, hepatocellular damage, and fibrosis. Triggering of inflammation in NASH is a key event in the progression of the disease, and identifying the factors that initiate or dysregulate this process is needed to develop strategies for its prevention or treatment. B cells have been implicated in several autoimmune and inflammatory diseases. However, their role in the pathogenesis of NAFLD and NASH is less clear. This review discusses the emerging evidence implicating intrahepatic B cells in the progression of NAFLD. We highlight the potential mechanisms of B-cell activation during NAFLD, such as increased hepatic expression of B-cell-activating factor, augmented oxidative stress, and translocation of gut-derived microbial products. We discuss the possible effector functions by which B cells promote NAFLD, including the production of proinflammatory cytokines and regulation of intrahepatic T cells and macrophages. Finally, we highlight the role of regulatory and IgA+ B cells in the pathogenesis of NASH-associated HCC. In this review, we make the case that future research is needed to investigate the potential of B-cell-targeting strategies for the treatment of NAFLD.

Microbiota-Driven Activation of Intrahepatic B Cells Aggravates NASH Through Innate and Adaptive Signaling.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis is rapidly becoming the leading cause of liver failure and indication for liver transplantation. Hepatic inflammation is a key feature of NASH but the immune pathways involved in this process are poorly understood. B lymphocytes are cells of the adaptive immune system that are critical regulators of immune responses. However, the role of B cells in the pathogenesis of NASH and the potential mechanisms leading to their activation in the liver are unclear. APPROACH AND RESULTS: In this study, we report that NASH livers accumulate B cells with elevated pro-inflammatory cytokine secretion and antigen-presentation ability. Single-cell and bulk RNA sequencing of intrahepatic B cells from mice with NASH unveiled a transcriptional landscape that reflects their pro-inflammatory function. Accordingly, B-cell deficiency ameliorated NASH progression, and adoptively transferring B cells from NASH livers recapitulates the disease. Mechanistically, B-cell activation during NASH involves signaling through the innate adaptor myeloid differentiation primary response protein 88 (MyD88) as B cell-specific deletion of MyD88 reduced hepatic T cell-mediated inflammation and fibrosis, but not steatosis. In addition, activation of intrahepatic B cells implicates B cell-receptor signaling, delineating a synergy between innate and adaptive mechanisms of antigen recognition. Furthermore, fecal microbiota transplantation of human NAFLD gut microbiotas into recipient mice promoted the progression of NASH by increasing the accumulation and activation of intrahepatic B cells, suggesting that gut microbial factors drive the pathogenic function of B cells during NASH. CONCLUSION: Our findings reveal that a gut microbiota-driven activation of intrahepatic B cells leads to hepatic inflammation and fibrosis during the progression of NASH through innate and adaptive immune mechanisms.

DC corral trapping of single nanoparticles and macromolecules in solution.

Progress in sorting, separating, and characterizing ever smaller amounts of chemical and biological material depends on the availability of methods for the controlled interaction with nanoscale and molecular-size objects. Here, we report on the reversible, tunable trapping of single DNA molecules and other charged micro- and nanoparticles in aqueous solution using a direct-current (DC) corral trap setup. The trap consists of a circular, non-conductive void in a metal-coated surface that, when charged, generates an electrostatic potential well in the proximate solution. Our results demonstrate that stable, nanoscale confinement of charged objects is achievable over extended periods of time, that trap stiffness is controlled by the applied voltage, and that simultaneous trapping of multiple objects is feasible. The approach shows great promise for lab-on-a-chip systems and biomedical applications due to its simplicity, scalability, selectivity, and the capability to manipulate single DNA molecules in standard buffer solutions.

Hepatic lysosomal acid lipase overexpression worsens hepatic inflammation in mice fed a Western diet.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets in hepatocytes. NAFLD development and progression is associated with an increase in hepatic cholesterol levels and decreased autophagy and lipophagy flux. Previous studies have shown that the expression of lysosomal acid lipase (LAL), encoded by the gene LIPA, which can hydrolyze both triglyceride and cholesteryl esters, is inversely correlated with the severity of NAFLD. In addition, ablation of LAL activity results in profound NAFLD. Based on this, we predicted that overexpressing LIPA in the livers of mice fed a Western diet would prevent the development of NAFLD. As expected, mice fed the Western diet exhibited numerous markers of NAFLD, including hepatomegaly, lipid accumulation, and inflammation. Unexpectedly, LAL overexpression did not attenuate steatosis and had only minor effects on neutral lipid composition. However, LAL overexpression exacerbated inflammatory gene expression and infiltration of immune cells in mice fed the Western diet. LAL overexpression also resulted in abnormal phagosome accumulation and lysosomal lipid accumulation depending upon the dietary treatment. Overall, we found that hepatic overexpression of LAL drove immune cell infiltration and inflammation and did not attenuate the development of NAFLD, suggesting that targeting LAL expression may not be a viable route to treat NAFLD in humans.

Environmentally Benign Carbon Nanodots Prepared from Lemon for the Sensitive and Selective Fluorescence Detection of Fe(III) and Tannic Acid.

Photoluminescent carbon nanodots (CNDs) were prepared using a biocarbon source of lemon extract. The obtained CNDs are of spherical shape and are enriched with the carboxylic acid fucntionalities. CNDs exhibited a fluorescence emission at 445 nm and unveiled blue luminescence in ultraviolet excitation. The influences of pH and ionic strength toward the stability of CNDs were investigated in detail and the obtained stability authenticates their applicability in different environmental conditions. The competitive binding of Fe3+ with CNDs quenches the fluorescence behavior of CNDs and was further quenched with the selective complex formation of Fe3+ with tannic acid (TA). The interference experiments specified that CNDs-Fe3+ assembly selectively detected TA and the co-existing molecules have not influenced the quenching effect of TA with CNDs-Fe3+. The analytical reliability of constructed sensor was validated from the recovery obtained in the range of 91.66-107.02% in real samples. Thus the low cost and environmentally benign CNDs prepared from natural biomass provide new avenues in the fluorescence detection of biologically significant metal ions and biomolecules, facilitating their competency in on-site applications of real environmental samples.

Acute lower gastrointestinal bleeding: are STRATE and BLEED scores valid in clinical practice?

AIM: Acute lower gastrointestinal bleeding (ALGIB) is a common gastroenterological emergency. Despite this, outcomes are difficult to predict. Only STRATE and BLEED scores are readily available. The aim of this study was to identify risk factors for severe ALGIB and access the validity of available scores. METHODS: Emergency consecutive admissions for ALGIB in the last 7 years were retrospectively reviewed. Severe ALGIB was defined as transfusion of ≥ 2 units of blood and/or a haematocrit decrease of ≥ 20% within the first 24 h and/or recurrent bleeding after 24 h of stability. STRATE and BLEED scores were calculated and concordance between score and respective severity outcome was accessed. RESULTS: A total of 132 patients were identified. Bleeding presented as haematochezia in 94.7% and was severe in 36.4% of cases. ALGIB was significantly more severe in patients with lower systolic (P = 0.02) and diastolic blood pressures on admission (P < 0.01), lower admission haemoglobin (P < 0.01), diverticular bleeding (P < 0.01), angioectasias (P = 0.02) and radiation colitis (P < 0.02). Severe ALGIB was associated with admission intermediate care units (P < 0.01), increased length of stay (P < 0.01) and volume of blood transfused (P < 0.01). We identified no significant association between outcomes with either the STRATE (P = 0.72) or BLEED scores (P = 0.05). CONCLUSIONS: Severe ALGIB is associated with greater length of stay, volume of blood transfusions and admission to greater level of care units. In our cohort, STRATE and BLEED scores were poor predictors of outcome.

Impact of infected and noninfected human dentine debris on bone healing in rats.

AIM: To evaluate in vivo the bone tissue response of rats to varying amounts of infected and noninfected dentine debris. METHODOLOGY: Bone tissue reactions were evaluated histologically in 42 Wistar rats after 7, 30 and 60 days. For each animal, three surgical cavities were prepared on the femur and filled with varying amounts (5, 10 or 20 mg) of infected or noninfected dentine debris pellets. In the negative control group, the surgical cavities were not filled. At the end of each experimental period, the animals were euthanized. The samples were processed histologically and analysed using a light microscope. The presence and the severity of inflammatory reaction, as well as hard tissue deposition were evaluated. Data were subjected to statistical analysis and the effects of the dependent variables calculated using nonparametric tests Kruskal-Wallis and Mann-Whitney U with due Bonferroni corrections at P = 0.05. RESULTS: At 7 days, the presence of infected debris significantly increased the histopathological scores for neutrophils (P < 0.05), and abscess formation (P < 0.05). Noninfected debris scored significantly higher for lymphocyte infiltrate compared with the control group and infected debris (P < 0.05). Both infected and noninfected debris equally triggered eosinophil cells compared with no-dentine (P < 0.05). As for giant cells and macrophages, no difference was detected amongst the dentine groups (P > 0.05). Hard tissue deposition was similar regardless of the presence or the bacteriological status of the dentine (P = 1.00). None of the above histopathological parameters was significantly influenced by the amount of debris (P > 0.05). For all parameters evaluated, at 7 days of analysis, the inflammatory response was significantly more intense compared with 30 and 60 days (P < 0.05). Inflammatory parameters were scored similarly for the evaluated groups after 30 and 60 days (P > 0.05). However, hard tissue deposition has significantly increased after 30 days (P < 0.05). No difference was seen between 30 and 60 days of analysis (P = 1.00) for all histological parameters evaluated. CONCLUSION: The assumption that the amount of extruded debris may negatively affect the inflammatory response of bone tissue was not validated in the present in vivo animal study. Infected dentine may trigger acute inflammatory parameters especially during the first 7 days of contact with the tissue; however, in the long term, these negative effects are mitigated.

Two-dimensional speckle tracking echocardiography demonstrates no effect of active acromegaly on left ventricular strain.

BACKGROUND: Speckle tracking echocardiography (STE) allows for the study of myocardial strain (ε), a marker of early and subclinical ventricular systolic dysfunction. Cardiac disease may be present in patients with acromegaly; however, STE has never been used to evaluate these patients. OBJECTIVE: To evaluate left ventricular (LV) global longitudinal strain in patients with active acromegaly with normal LV systolic function. DESIGN: Cross-sectional clinical study. METHODS: Patients with active acromegaly with no detectable heart disease and a control group were matched for age, gender, arterial hypertension and diabetes mellitus underwent STE. Global LV longitudinal ε (GLS), left ventricular mass index (LVMi), left ventricular ejection fraction (LVEF) and relative wall thickness (RWT) were obtained via two-dimensional (2D) echocardiography using STE. RESULTS: Thirty-seven patients with active acromegaly (mean age 45.6 ± 13.8; 48.6% were males) and 48 controls were included. The mean GLS was not significantly different between the acromegaly group and the control group (in %, -20.1 ± 3.1 vs. -19.4 ± 2.2, p = 0.256). Mean LVMi was increased in the acromegaly group (in g/m2, 101.6 ± 27.1 vs. 73.2 ± 18.6, p < 0.01). There was a negative correlation between LVMi and GLS (r = -0.39, p = 0.01). CONCLUSIONS: Acromegaly patients, despite presenting with a higher LVMi when analyzed by 2D echocardiography, did not present with impairment in the strain when compared to a control group; this finding indicates a low chance of evolution to systolic dysfunction and agrees with recent studies that show a lower frequency of cardiac disease in these patients.

State-Specific Reactions of Cu(+)((1)S,(3)D) with SF6 and SF5Cl.

State-specific reactions of Cu(+)((1)S,(3)D) were carried out in a selected ion drift cell apparatus with SF6 and SF5Cl. Copper ions were prepared in a glow discharge utilizing Ne as the working gas. Analysis of Cu(+) states using ion mobility mass spectrometry (IMS) indicated the presence of both Cu(+)(3d(10)) and Cu(+)(3d(9)4s(1)) configurations attributable to the (1)S ground and (3)D first excited states of this metal ion, respectively. State-specific product formation in reactions of these ions with the two neutral substrates of interest here was determined using IMS along with both known and calculated energetic requirements for product formation. These experiments indicate that Cu(+)((1)S) associates with both SF6 and SF5Cl; however, the process is approximately four times as efficient with the latter neutral under these conditions. Association is also observed as a minor product between Cu(+)((3)D) and both neutral reactants. Inefficient formation of SF3(+) occurs as the sole bimolecular product from SF6 via Cu(+)((3)D). In contrast, Cu(+)((3)D) reacts with SF5Cl in rapid parallel bimolecular processes yielding SF3(+) and CuCl(+). These results also indicate that CuCl(+) initiates additional higher-order processes which result in SF5(+) and SF4Cl(+). The energetics associated with the formation of SF3(+) suggest that a copper halide neutral byproduct must also be formed, requiring a more complex mechanism than simple dissociative charge-transfer.

State-specific reactions of Cu(+)((1)S,(3)D,(1)D) with the super greenhouse gas SF5CF3.

State-specific reactions of the potent greenhouse gas SF5CF3 with Cu(+) were carried out in a selected ion drift cell apparatus. Copper ions were prepared in a glow discharge utilizing Ne as the working gas. Analysis of these ions using ion mobility mass spectrometry (IMS) indicated the presence of both Cu(+)(3d(10)) and Cu(+)(3d(9)4s(1)) configurations. Subsequent analysis indicates that the 3d(10) configuration consists of Cu(+)((1)S) exclusively whereas the 3d(9)4s(1)configuration is composed primarily of Cu(+)((3)D) with small contributions from Cu(+)((1)D). State-specific product formation in reactions of these ions with SF5CF3 was determined using IMS along with the known energetic requirements for product formation. These experiments reveal that Cu(+) excited states initiate fragmentation of SF5CF3 to yield SF2(+), SF3(+), SF5(+), and CF3(+), where SF3(+) represents the largest branching fraction at 90% of the total bimolecular product formation. The energetics associated with the formation of these ions suggest that molecular Cu-containing products must also be formed in all cases, indicating that the governing reaction mechanisms are more complicated than simple dissociative charge transfer. Production of SF2(+) and SF3(+) are shown to proceed via Cu(+)((3)D) and can be rationalized with a two-step mechanism proceeding through the common intermediate SF3CF3(+). Production of CF3(+) can be explained using this same mechanism but is also energetically possible from Cu(+)((1)D) in a more direct process. Energetic requirements indicate that Cu(+)((1)D) is the sole source of SF5(+) with concomitant formation of CuCF3. Cu(+)((1)S) exhibits adduct formation exclusively, but IMS spectra of the resulting Cu(+)·SF5CF3 suggest that as many as three association structures are formed.

Mandibular symphysis and ramus as sources of osteoblastic cells for bone tissue engineering.

OBJECTIVES: Autografts from mandibular symphysis and ramus are often used for bone reconstruction. Based on this, we hypothesized that these sites could be useful cell sources for bone tissue engineering approaches. Thus, our study aimed at evaluating the proliferation and osteoblast phenotype development of cells derived from mandibular symphysis and ramus. MATERIALS AND METHODS: Cells were isolated from bone fragments of four patients by enzymatic digestion and cultured under osteogenic condition for up to 17 days. Cultures were assayed for cell proliferation, gene expression of key bone markers runt-related transcription factor 2 (Runx2), distal-less homeobox 5 (DLX5), SATB homeobox 2 (SATB2), Osterix (OSX), family with sequence similarity 20, member C (FAM20C), bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), alkaline phosphatase (ALP) expression and activity, and extracellular matrix mineralization. Data were compared by two-way ANOVA or t-test for independent samples when appropriate. RESULTS: Cells derived from ramus displayed lower proliferative activity and higher gene expression of Runx2, DLX5, SATB2, OSX, FAM20C, BSP, OPN and OC, ALP protein expression and activity and extracellular matrix mineralization compared with symphysis-derived cells. CONCLUSION: Symphysis and ramus may be considered as cell sources for bone tissue engineering approaches but due to the higher osteogenic potential, ramus-derived cells are more appealing for constructing cell-based biomaterials.

2-Amino-5-nitro-pyridinium hydrogen oxalate.

In the cation of the title mol-ecular salt, C5H6N3O2 (+)·C2HO4 (-), the dihedral angle between the aromatic ring and the nitro group is 3.5 (3)°; in the anion, the dihedral angle between the CO2 and CO2H planes is 10.5 (2)°. In the crystal, the anions are linked into [100] chains by O-H⋯O hydrogen bonds. The cations cross-link the chains by way of N-H⋯O hydrogen bonds and the structure is consolidated by C-H⋯O inter-actions.

Nerves in quadratus lumborum planes: A cadaveric study.

Dermatomal analgesia achieved with quadratus lumborum blocks is site-dependent and inconsistent. Cadaveric and clinical studies reveal multiple mechanisms of action. We dissected six fresh human cadavers bilaterally and thoroughly studied their neurological linkages to the quadratus lumborum muscle (QLM) to identify neural structures and block targets. At the end of the investigation, only the subcostal nerve (anterolateral) and the ilioinguinal nerves were found near the QLM in all specimens. The iliohypogastric nerve was found in only two specimens. No further neural targets were found in the fascial planes before and posterior to the QLM.