Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

The phageome in normal and inflamed human skin.

Dysbiosis of skin microbiota drives the progression of atopic dermatitis (AD). The contribution of bacteriophages to bacterial community compositions in normal and inflamed skin is unknown. Using shotgun metagenomics from skin swabs of healthy individuals and patients with AD, we found 13,586 potential viral contiguous DNA sequences, which could be combined into 164 putative viral genomes including 133 putative phages. The Shannon diversity index for the viral metagenome-assembled genomes (vMAGs) did not correlate with AD. In total, we identified 28 vMAGs that differed significantly between normal and AD skin. Quantitative polymerase chain reaction validation of three complete vMAGs revealed their independence from host bacterium abundance. Our data indicate that normal and inflamed skin harbor distinct phageomes and suggest a causative relationship between changing viral and bacterial communities as a driver of skin pathology.

Comparison of surgical techniques for optimal lead placement in sacral neuromodulation: a cadaver study.

BACKGROUND: Sacral neuromodulation (SNM) is a common treatment for patients with urinary and faecal incontinence. A close contact of the tined lead electrode with the targeted nerve is likely to improve functional outcome. The aim of this study was to compare the position of the SNM lead in relation to the sacral nerve by comparing different implantation techniques. METHODS: This cadaver study was conducted at the Division of Anatomy of Vienna's Medical University in October 2020. We dissected 10 cadavers after bilateral SNM lead implantation (n = 20), using two different standardized implantation techniques. The cadavers were categorized as group A (n = 10), representing the conventional guided implantation group and group B (n = 10), where SNM implantation was conducted with the novel fluoroscopy-guided "H"-technique. The primary goal was to assess the distance between the sacral nerve and the lead placement. RESULTS: The electrodes were inserted at a median angle of 58.5° (46-65°) in group A and 60° (50-65°) in group B, without reaching statistical significance. In 8 cadavers, the lead entered the S3 foramen successfully. The median distance of the lead to the nerve did not show a significant difference between both groups (E0: Group A: 0.0 mm vs. Group B: 0.0 mm, p = 0.969; E1: Group A: 0.0 mm vs. Group B: 0.5 mm p = 0.754; E2: Group A: 2.5 mm vs. Group B: 2.5 mm p = 1.000; E3: Group A: 3.5 mm vs. Group B: 4.0 mm p = 0.675). In 2 cases (20%) of the conventional group A, the lead was misplaced and located at the gluteal muscle. Perforation of the presacral fascia was observed in one lead placement in group A and in two placements in group B. CONCLUSIONS: Both standardized implantation techniques may ensure close electrode proximity to the targeted nerve. Misplacement of the electrode was more often observed with the conventional implantation technique.

Ultrasound for the detection of the pyramidal lobe of the thyroid gland.

PURPOSE: The pyramidal lobe (PL) is an ancillary lobe of the thyroid gland that can be affected by the same pathologies as the rest of the gland. We aimed to assess the diagnostic performance of high-resolution sonography in the detection of the PL with verification by dissection and histological examination. METHODS: In a prospective, cross-sectional mono-center study, 50 fresh, non-embalmed cadavers were included. Blinded ultrasound examination was performed to detect the PL by two investigators of different experience levels. If the PL was detected with ultrasound, dissection was performed to expose the PL and obtain a tissue sample. When no PL was detected with ultrasound, a tissue block of the anterior cervical region was excised. An endocrine pathologist microscopically examined all tissue samples and tissue blocks for the presence of thyroid parenchyma. RESULTS: The prevalence of the PL was 80% [40/50; 95% CI (68.9%; 91.1%)]. Diagnostic performance for both examiners was: sensitivity (85.0%; 42.5%), specificity (50.0%; 60.0%), positive predictive value (87.2%; 81.0%), negative predictive value (45.5%; 21.0%) and accuracy (78.0%; 46.0%). Regression analysis demonstrated that neither thyroid parenchyma echogenicity, thyroid gland volume, age nor body size proved to be covariates in the accurate detection of a PL (p > .05). CONCLUSION: We report that high-resolution ultrasound is an adequate examination modality to detect the PL. Our findings indicate a higher prevalence than previously reported. Therefore, the PL may be regarded as a regular part of the thyroid gland. We also advocate a dedicated assessment of the PL in routine thyroid ultrasound.

A case of COVID-19 vaccination-associated forme fruste purpura fulminans.

We report the case of a female, 77 year old patient with multi-localized skin infarctions following vaccination with mRNA-1273 (Moderna). This phenomenon is to our knowledge otherwise only seen in infection-associated purpura fulminans - which was thoroughly ruled out in our patient. This report demonstrates that we need to be vigilant of a wider array of vascular phenomena related to Covid vaccinations.

Single-cell RNA sequencing profiling in a patient with discordant primary cutaneous B-cell and T-cell lymphoma reveals micromilieu-driven immune skewing.

BACKGROUND: Primary cutaneous lymphomas comprise a heterogeneous group of B-cell and T-cell malignancies which often show an indolent course, but can progress to aggressive disease in a subset of patients. Diagnosis is often delayed owing to clinical and histopathological similarities with benign inflammatory conditions. Especially during early disease, cancer cells are present at relatively low percentages compared with the inflammatory infiltrate, an interplay that is currently only insufficiently understood. OBJECTIVES: To improve diagnostics and perform molecular characterization of a complex type of primary cutaneous lymphoma. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed and combined with T-cell and B-cell receptor sequencing. RESULTS: We were able to diagnose a patient with concurrent mycosis fungoides (MF) and primary cutaneous follicle centre lymphoma (PCFCL), appearing in mutually exclusive skin lesions. Profiling of tumour cells and the tissue microenvironment revealed a type-2 immune skewing in MF, most likely guided by the expanded clone that also harboured upregulation of numerous pro-oncogenic genes. By contrast, PCFCL lesions exhibited a more type-1 immune phenotype, consistent with its indolent behaviour. CONCLUSIONS: These data not only illustrate the diagnostic potential of scRNA-seq, but also allow the characterization of specific clonal populations that shape the unique tissue microenvironment in clinically distinct types of lymphoma skin lesions.

Standardized fluoroscopy-guided implantation technique enables optimal electrode placement in sacral neuromodulation: a cadaver study.

BACKGROUND: Sacral neuromodulation (SNM) is an established treatment option for patients with faecal incontinence. The location of the stimulating electrode is considered to be essential for treatment success. The purpose of this study was to evaluate the position of SNM electrodes after using a standardized fluoroscopy-guided implantation technique. METHODS: For this cadaver study, SNM electrodes were implanted bilaterally in 5 lower body specimens. The lower edge of the sacroiliac joint and the medial edge of the sacral foramina were marked using fluoroscopy to draw an 'H' with the crossing points identifying S3. After electrode placement the pelvis was dissected to describe the exact position of the SNM electrodes. RESULTS: The electrodes were inserted at an angle with a median degree measure of 60° (range 50-65°) to the skin, with a median distance of 9 mm (range 0-13 mm) from the S3 marking. All electrodes entered the third sacral foramen. The median distance of the electrodes to the sacral nerve was 0 mm (range 0-3 mm) for the most proximal, 0.5 mm (range 0-5 mm) for the second, 2.25 mm (range 0-11 mm) for the third and 1.75 mm (range 0-16 mm) for the most distant electrode. There was neither a significant difference in the proximity of the electrodes to the nerve between the right and left side (proximal to distal electrode: p = 0.18, p = 0.16, p = 0.07, p = 0.07) nor between male and female cadavers (p = 0.25, p = 0.21, p = 0.66, p = 0.66). CONCLUSIONS: A standardized fluoroscopy-guided implantation technique enables a close contact between electrode and nerve. This can potentially result in an improved clinical outcome.

Neutrophil gelatinase associated lipocalin (NGAL) is elevated in type 2 diabetics with carotid artery stenosis and reduced under metformin treatment.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), an acute phase protein released by neutrophils, has been described as biomarker of inflammatory states. Type 2 diabetes mellitus (T2DM) is characterized by increased inflammation and an elevated risk for embolization of carotid artery stenosis (CAS). We aimed to explore the role of NGAL systemically and in plaques of diabetics undergoing carotid endarterectomy. Moreover, the potential anti-inflammatory effect of metformin on NGAL was addressed in diabetics. METHODS: Serum NGAL and matrix metalloproteinase (MMP)-9/NGAL levels were measured in 136 patients (67 with T2DM vs. 69 non-diabetics) by specific ELISA. Endarterectomy samples were graded histologically according to the American Heart Association´s classification. NGAL mRNA expression was detected using RealTime-PCR in carotid endarterectomy specimens. RESULTS: Serum NGAL [median 107.4 ng/ml (quartiles: 75.2-145.0) vs. 64.4 (50.4 -81.3), p < 0.0001] and MMP-9/NGAL [41.5 ng/ml (20.8-63.9) vs. 27.6 (16.0-42.4), p = 0.017] were significantly elevated in diabetics compared to non-diabetics, as were leukocytes, neutrophils, C-reactive protein and fibrinogen (all p < 0.05). In patients with symptomatic and asymptomatic CAS diabetics had higher NGAL levels compared to non-diabetics [128.8 ng/ml (100.8-195.6) vs. 64.8 (48.9-82.2] and [101.6 ng/ml (70.1-125.3) vs. 63.8 (51.0-81.3), respectively, both p < 0.0001]. Presence of T2DM and type VI plaques (with surface defect, hemorrhage or thrombus) had a profound impact on NGAL levels (both p < 0.01) in multiple linear regression analysis. NGAL mRNA was detectable in 95% of analyzed carotid artery lesions of diabetics compared to 5% of non-diabetics (p < 0.0001). Accordingly, cerebral embolization was more frequent in diabetics (52.2% vs. 29%, p = 0.006). Metformin treatment was associated with decreased NGAL [60.7 ng/ml (51.9-69.2) vs. 121.7 (103.7-169.9), p < 0.0001] and MMP-9/NGAL [20.8 ng/ml (12.1-26.5) vs. 53.7 (27.4-73.4), p = 0.007] in diabetics and reduced leukocyte infiltration in carotid lesions of diabetics. CONCLUSIONS: Higher NGAL levels in serum and plaques are associated with T2DM in patients with CAS. Metformin significantly reduced the inflammatory burden including NGAL in diabetics. Early treatment of these patients may be recommended, as elevated NGAL levels were linked with vulnerable plaques prone for embolization.

Serpinb9 is a marker of antigen cross-presenting dendritic cells.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

Ultrasound of the Hypoglossal Nerve in the Neck: Visualization and Initial Clinical Experience with Patients.

BACKGROUND AND PURPOSE: The hypoglossal nerve, providing motor innervation for the tongue, can be affected in many diseases of the neck and skull base, leading to dysarthria, dysphagia, and ultimately atrophy of the tongue. We determined the feasibility of direct visualization of the hypoglossal nerve in the neck with ultrasound, testing this technique on healthy volunteers and evaluating it in clinical practice. MATERIALS AND METHODS: The study consisted of 4 parts: first, ultrasound-guided perineural ink injections along the course of the hypoglossal nerve at 24 sides of 12 fresh, nonembalmed cadaver necks. Subsequently, the specimens were dissected to confirm the correct identification of the nerve. The second part was examination of healthy volunteers with ultrasound and measurement of cross-sectional areas for generating reference data. The third part was scanning of healthy volunteers by 2 resident physicians with little and intermediate experience in ultrasound. Fourth was examination with ultrasound of patients with motor symptoms of the tongue. RESULTS: The hypoglossal nerve was correctly identified bilaterally in all cadaveric specimens (24/24) and all volunteers (33/33). The cross-sectional area ranged from 1.9 to 2.1 mm(2). The resident physicians were able to locate the nerve in 19 of 22 cases, demonstrating that locating the nerve is reproducible and feasible even with intermediate experience in ultrasound. Finally, alterations of the hypoglossal nerve in disease states could be depicted. CONCLUSIONS: Direct, reliable, and reproducible visualization of the extracranial hypoglossal nerve with ultrasound is feasible.

The embryogenesis of the equine femorotibial joint: The equine interzone.

REASONS FOR PERFORMING STUDY: Articular cartilage regeneration is the focus and goal of considerable research effort. Since articular chondrocytes descend from a distinct cohort of progenitor cells located in embryonic nascent joints (interzones), establishing the timing of equine interzone formation is an essential first step towards understanding equine joint and articular cartilage development. OBJECTIVES: To establish the time frame during which the equine femorotibial interzone forms. STUDY DESIGN: Descriptive anatomical study. METHODS: Equine embryos were harvested at 37 (E37), 40, 42, 45, 50 and 65 days' gestation. The femorotibial interzone was examined using high-resolution episcopic microscopy of E37, E42, E45, E50 and E65. Additional histology and collagen-II-immunohistochemistry were performed on E42. RESULTS: At E37, the femorotibial interzone is first visible as a uniform layer, while at E42 the interzone is fully formed and consists of 3 morphologically distinct layers. The first evidence of cavitation was seen at E45. At E50, the cruciate ligaments were well formed and by E65, joint formation appeared complete. CONCLUSIONS: The embryogenesis of the equine femorotibial joint is similar to the developmental timeline of stage-matched human and murine embryos. Further studies looking at interzone formation on a cellular and molecular level may further our understanding of the intricate developmental patterns and pathways of articular cartilage development.

The dermal arteries of the human thumb pad.

The arteries of the skin have been postulated to form a profound plexus at the dermal/hypodermal junction and a superficial plexus in the papillary dermis. Our article aims to rebut this concept and to provide an alternative description of the arrangement of the dermal arteries. Employing a novel technique, we produced digital volume data (volume size: 2739 × 2054 × 3000 µm(3) ; voxel size: 1.07 × 1.07 × 2 µm(3) ) from biopsies of the skin of the thumb pads of 15 body donors. Utilizing these data, we analysed the arrangement of the dermal arteries with the aid of virtual re-sectioning tools, and, in three specimens, with high-quality three-dimensional (3D) surface models. In all specimens we observed a tree-like ramification of discrete dermal arteries. The terminal branches of the arterial trees gave rise to the ascending segments of the capillary loops of the dermal papillae. None of the specimens showed a superficial arterial plexus. This suggests that the skin of the human thumb pad can be split in discrete 'arterial units'. Each unit represents the zone of the papillary dermis and epidermal/dermal junction, to which blood is supplied exclusively by the branches of a single dermal artery. The concept of dermal arterial units is in contrast to all existing descriptions of the architecture of the dermal arteries. However, whether it can be transferred to the skin of other body parts, remains to be tested. Likewise, the consequences of arterial units for understanding the mechanisms of wound healing and the appearance and genesis of skin diseases remain to be examined.

A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy.

BACKGROUND: Multiphoton microscopy (MPM) is a novel imaging technology that has recently become applicable for diagnostic purposes. The use of (near) infrared light in MPM allows for deep tissue imaging. In addition, this modality exploits the autofluorescent nature of extracellular matrix fibres within the skin. OBJECTIVES: To quantitate the structure and abundance of elastic fibres in human dermis in three dimensions utilizing autofluorescent signals generated by MPM for the objective examination of elastin-related skin disorders. METHODS: Cross-sections of skin samples from elastin-related disorders were analysed by MPM and correlated to histopathology. In situ visualization of elastic fibres by MPM was conducted by en face imaging of ex vivo skin samples through the intact epidermis. Image analysis software was used to quantify elastic fibres in three dimensions. RESULTS: Based on the MPM-detected elastin-specific autofluorescence, we developed the Dermal Elastin Morphology Index (DEMI), calculated as the ratio of elastic fibre surface area and volume. This enabled objective three-dimensional quantification of elastic fibres. Quantitative scoring of sun-damaged skin using DEMI correlated with qualitative histopathological grading of the severity of solar elastosis. Furthermore, this approach was applied to changes in elastic fibre architecture in other disorders, such as pseudoxanthoma elasticum (PXE), PXE-like syndrome, elastofibroma, focal dermal elastosis, anetoderma, mid-dermal elastolysis and striae distensae. We imaged elastic fibres in intact ex vivo skin imaged en face through the epidermis, indicating that this approach could be used in vivo. CONCLUSIONS: MPM has the potential for noninvasive in vivo visualization of elastic fibres in the dermis with near histological resolution. DEMI allows objective assessment of elastic fibres to support diagnosis and monitoring of disease progress or therapy of elastin-related skin disorders.

A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis.

Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis.

Ret finger protein inhibits muscle differentiation by modulating serum response factor and enhancer of polycomb1.

Skeletal myogenesis is precisely regulated by multiple transcription factors. Previously, we demonstrated that enhancer of polycomb 1 (Epc1) induces skeletal muscle differentiation by potentiating serum response factor (SRF)-dependent muscle gene activation. Here, we report that an interacting partner of Epc1, ret finger protein (RFP), blocks skeletal muscle differentiation. Our findings show that RFP was highly expressed in skeletal muscles and was downregulated during myoblast differentiation. Forced expression of RFP delayed myoblast differentiation, whereas knockdown enhanced it. Epc1-induced enhancements of SRF-dependent multinucleation, transactivation of the skeletal α-actin promoter, binding of SRF to the serum response element, and muscle-specific gene induction were blocked by RFP. RFP interfered with the physical interaction between Epc1 and SRF. Muscles from rfp knockout mice (Rfp(-/-)) mice were bigger than those from wild-type mice, and the expression of SRF-dependent muscle-specific genes was upregulated. Myotube formation and myoblast differentiation were enhanced in Rfp(-/-) mice. Taken together, our findings highlight RFP as a novel regulator of muscle differentiation that acts by modulating the expression of SRF-dependent skeletal muscle-specific genes.

Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos.

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 mum x 2 mum x 2 mum and 3 mum x 3 mum x 3 mum) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.

[Morphology and functional anatomy of the growing thorax]. / Morphologie und funktionelle Anatomie des wachsenden Thorax.

Morphogenesis of most thoracic organs and structures is not finished with birth but perpetuates in postnatal life. The postnatal growth is partly associated with enormous changes in structure, morphology, and function. Our study presents an overview of morphological, topological and functional peculiarities of thoracic anatomy during infancy, childhood and adolescence. It focuses on the development of the mammary gland, osseous structures of the chest wall, thymus, heart, and lungs. Most of the presented data are based on post-mortem studies. Measurements and numerical data are mainly included for illustration of growth-associated changes.