Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Predictive molecular pathology after prolonged fixation: A study on tissue from anatomical body donors.

Histopathological assessment of tissue samples after prolonged formalin fixation has been described previously, but currently there is only limited knowledge regarding the feasibility of molecular pathology on such tissue. In this pilot study, we tested routine molecular pathology methods (DNA isolation, DNA pyrosequencing/next-generation sequencing, DNA methylation analysis, RT-PCR, clonality analysis and fluorescence in situ hybridization) on tissue samples from 11 tumor entities as well as non-neoplastic brain tissue from 43 body donors during the gross anatomy course at Ulm University (winter semester 2019/20 and 2020/21). The mean post mortem interval until fixation was 2.5 ± 1.6 days (range, 1-6 days). Fixation was performed with aqueous formaldehyde solution (formalin, 1.5-2%). The mean storage time of body donors was 12.8 ± 5.6 months (range, 7-25 months). While most diagnostic methods were successful, samples showed significant variability in DNA quality and evaluability. DNA pyrosequencing as well as next-generation sequencing was successful in all investigated samples. Methylation analyses were partially not successful in some extend due to limited intact DNA yield for these analyses. Taken together, the use of prolonged formalin-fixed tissue samples from body donors offers new avenues in research and education, as these samples could be used for morpho-molecular studies and the establishment of biobanks, especially for tissue types that cannot be preserved and studied in vivo. Pathological ward rounds, sample collection, and histopathological and molecular workup have been integrated in the gross anatomy course in Ulm as an integral part of the curriculum, linking anatomy and pathology and providing medical students early insight into the broad field of (molecular) pathology.

Combined expansion and STED microscopy reveals altered fingerprints of postsynaptic nanostructure across brain regions in ASD-related SHANK3-deficiency.

Synaptic dysfunction is a key feature of SHANK-associated disorders such as autism spectrum disorder, schizophrenia, and Phelan-McDermid syndrome. Since detailed knowledge of their effect on synaptic nanostructure remains limited, we aimed to investigate such alterations in ex11|SH3 SHANK3-KO mice combining expansion and STED microscopy. This enabled high-resolution imaging of mosaic-like arrangements formed by synaptic proteins in both human and murine brain tissue. We found distinct shape-profiles as fingerprints of the murine postsynaptic scaffold across brain regions and genotypes, as well as alterations in the spatial and molecular organization of subsynaptic domains under SHANK3-deficient conditions. These results provide insights into synaptic nanostructure in situ and advance our understanding of molecular mechanisms underlying synaptic dysfunction in neuropsychiatric disorders.

Lost in space: what single-cell RNA sequencing cannot tell you.

Plant scientists are rapidly integrating single-cell RNA sequencing (scRNA-seq) into their workflows. Maximizing the potential of scRNA-seq requires a proper understanding of the spatiotemporal context of cells. However, positional information is inherently lost during scRNA-seq, limiting its potential to characterize complex biological systems. In this review we highlight how current single-cell analysis pipelines cannot completely recover spatial information, which confounds biological interpretation. Various strategies exist to identify the location of RNA, from classical RNA in situ hybridization to spatial transcriptomics. Herein we discuss the possibility of utilizing this spatial information to supervise single-cell analyses. An integrative approach will maximize the potential of each technology, and lead to insights which go beyond the capability of each individual technology.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Melanocyte Density in the Diagnosis of Melanoma In Situ in Sun-Damaged Skin.

ABSTRACT: Histologic differentiation between melanoma in situ in chronically sun-damaged skin (CSDS) [lentigo maligna (LM)] and CSDS without malignancy is difficult because signs of melanocyte activation and proliferation are found in both. A potentially reliable and quantifiable criterion is melanocyte density (MD). Here, we evaluated whether and to what extent MD allows the distinction between LM and CSDS, which is particularly relevant for the evaluation of borderline cases and surgical margins.Articles assessing MD in LM and/or CSDS were evaluated in a systematic review. The results were categorized and compared according to staining. Cutoff values were included whenever stated.Twenty articles matched the selection criteria. Six hundred forty-four samples of CSDS and 227 samples of LM were considered. In each individual study, mean MD scores were higher for LM than for CSDS. However, looking at the overall study situation, it becomes clear that the data are very heterogeneous and show overlaps. Therefore, no reliable orientation value can be derived. Only 1 article defined a cutoff value.The data of MD in LM in contrast to CSDS were sparse, and a defined cutoff value was only mentioned in 1 article for microphthalmia-associated transcription factor, which cannot yet be generalized. Especially regarding the importance for the definition of surgical resection margins, this unsatisfactory data set highlights the need for further studies. More precise diagnostic criteria could spare some patients extensive and possibly disfiguring surgery.

Disrupted extracellular matrix and cell cycle genes in autism-associated Shank3 deficiency are targeted by lithium.

The Shank3 gene encodes the major postsynaptic scaffolding protein SHANK3. Its mutation causes a syndromic form of autism spectrum disorder (ASD): Phelan-McDermid Syndrome (PMDS). It is characterized by global developmental delay, intellectual disorders (ID), ASD behavior, affective symptoms, as well as extra-cerebral symptoms. Although Shank3 deficiency causes a variety of molecular alterations, they do not suffice to explain all clinical aspects of this heterogenic syndrome. Since global gene expression alterations in Shank3 deficiency remain inadequately studied, we explored the transcriptome in vitro in primary hippocampal cells from Shank3∆11(-/-) mice, under control and lithium (Li) treatment conditions, and confirmed the findings in vivo. The Shank3∆11(-/-) genotype affected the overall transcriptome. Remarkably, extracellular matrix (ECM) and cell cycle transcriptional programs were disrupted. Accordingly, in the hippocampi of adolescent Shank3∆11(-/-) mice we found proteins of the collagen family and core cell cycle proteins downregulated. In vitro Li treatment of Shank3∆11(-/-) cells had a rescue-like effect on the ECM and cell cycle gene sets. Reversed ECM gene sets were part of a network, regulated by common transcription factors (TF) such as cAMP responsive element binding protein 1 (CREB1) and ß-Catenin (CTNNB1), which are known downstream effectors of synaptic activity and targets of Li. These TFs were less abundant and/or hypo-phosphorylated in hippocampi of Shank3∆11(-/-) mice and could be rescued with Li in vitro and in vivo. Our investigations suggest the ECM compartment and cell cycle genes as new players in the pathophysiology of Shank3 deficiency, and imply involvement of transcriptional regulators, which can be modulated by Li. This work supports Li as potential drug in the management of PMDS symptoms, where a Phase III study is ongoing.

The Role of Phosphatidylethanolamine N-Methyltransferase (PEMT) and Its Waist-Hip-Ratio-Associated Locus rs4646404 in Obesity-Related Metabolic Traits and Liver Disease.

In previous genome-wide association studies (GWAS), genetic loci associated with obesity and impaired fat distribution (FD) have been identified. In the present study, we elucidated the role of the PEMT gene, including the waist-hip-ratio-associated single nucleotide polymorphism rs4646404, and its influence on obesity-related metabolic traits. DNA from 2926 metabolically well-characterized subjects was used for genotyping. PEMT expression was analyzed in paired visceral (vis) and subcutaneous (sc) adipose tissue (AT) from a subset of 574 individuals. Additionally, PEMT expression was examined in vis, sc AT and liver tissue in a separate cohort of 64 patients with morbid obesity and liver disease. An in vitro Pemt knockdown was conducted in murine epididymal and inguinal adipocytes. Our findings highlight tissue-specific variations in PEMT mRNA expression across the three studied tissues. Specifically, vis PEMT mRNA levels correlated significantly with T2D and were implicated in the progression of non-alcoholic steatohepatitis (NASH), in contrast to liver tissue, where no significant associations were found. Moreover, sc PEMT expression showed significant correlations with several anthropometric- and metabolic-related parameters. The rs4646404 was associated with vis AT PEMT expression and also with diabetes-related traits. Our in vitro experiments supported the influence of PEMT on adipogenesis, emphasizing its role in AT biology. In summary, our data suggest that PEMT plays a role in regulating FD and has implications in metabolic diseases.

Expression profiles of the autism-related SHANK proteins in the human brain.

BACKGROUND: SHANKs are major scaffolding proteins at postsynaptic densities (PSDs) in the central nervous system. Mutations in all three family members have been associated with neurodevelopmental disorders such as autism spectrum disorders (ASDs). Despite the pathophysiological importance of SHANK2 and SHANK3 mutations in humans, research on the expression of these proteins is mostly based on rodent model organisms. RESULTS: In the present study, cellular and neuropil SHANK2 expression was analyzed by immunofluorescence (IF) staining of post mortem human brain tissue from four male individuals (19 brain regions). Mouse brains were analyzed in comparison to evaluate the degree of phylogenetic conservation. Furthermore, SHANK2 and SHANK3 isoform patterns were compared in human and mouse brain lysates. While isoform expression and subcellular distribution were largely conserved, differences in neuropil levels of SHANK2 were found by IF staining: Maximum expression was concordantly measured in the cerebellum; however, higher SHANK2 expression was detected in the human brainstem and thalamus when compared to mice. One of the lowest SHANK2 levels was found in the human amygdala, a moderately expressing region in mouse. Quantification of SHANK3 IF in mouse brains unveiled a distribution comparable to humans. CONCLUSIONS: In summary, these data show that the overall expression pattern of SHANK is largely conserved in defined brain regions; however, differences do exist, which need to be considered in the translation of rodent studies. The summarized expression patterns of SHANK2 and SHANK3 should serve as a reference for future studies.

A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation.

BACKGROUND: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Humoral and cellular immune responses in fully vaccinated individuals with or without SARS-CoV-2 breakthrough infection: Results from the CoV-ADAPT cohort.

Despite recent advances in prophylactic vaccination, SARS-CoV-2 infections continue to cause significant morbidity. A better understanding of immune response differences between vaccinated individuals with and without later SARS-CoV-2 breakthrough infection is urgently needed. CoV-ADAPT is a prospective long-term study comparing humoral (anti-spike-RBD-IgG, neutralization capacity, avidity) and cellular (spike-induced T-cell interferon-γ [IFN-γ] release) immune responses in individuals vaccinated against SARS-CoV-2 at four different time points (three before and one after third vaccination). In this cohort study, 62 fully vaccinated individuals presented with SARS-CoV-2 breakthrough infections vs 151 without infection 3-7 months following third vaccination. Breakthrough infections significantly increased anti-spike-RBD-IgG (p < 0.01), but not spike-directed T-cell IFN-γ release (TC) or antibody avidity. Despite comparable surrogate neutralization indices, the functional neutralization capacity against SARS-CoV-2-assessed via a tissue culture-based assay-was significantly higher following breakthrough vs no breakthrough infection. Anti-spike-RBD-IgG and antibody avidity decreased with age (p < 0.01) and females showed higher anti-spike-RBD-IgG (p < 0.01), and a tendency towards higher antibody avidity (p = 0.051). The association between humoral and cellular immune responses previously reported at various time points was lost in subjects after breakthrough infections (p = 0.807). Finally, a machine-learning approach based on our large immunological dataset (a total of 49 variables) from different time points was unable to predict breakthrough infections (area under the curve: 0.55). In conclusion, distinct differences in humoral vs cellular immune responses in fully vaccinated individuals with or without breakthrough infection could be demonstrated. Breakthrough infections predominantly drive the humoral response without boosting the cellular component. Breakthrough infections could not be predicted based on immunological data, which indicates a superior role of environmental factors (e.g., virus exposure) in individualized risk assessment.

Full-body blue light irradiation as treatment for atopic dermatitis: a randomized sham-controlled clinical trial (AD-Blue).

BACKGROUND: Visible blue light (wavelength 400-495 nm) is a promising new treatment option for both psoriasis and atopic dermatitis (AD). Whilst previous clinical trials featured various devices and blue light at a variety of wavelengths, none of these interventions were challenged in objective clinical criteria. PATIENTS AND METHODS: Eighty-seven patients diagnosed with AD were enrolled in AD-Blue, an international, prospective, double-blinded, three-armed (415 nm vs. 450 nm vs. sham control), randomized trial designed to investigate the safety and efficacy of prototype full-body blue light devices. RESULTS: Full-body irradiation with 450 nm blue light but not 415 nm had a significant impact on itch (Itch-VAS, -1.6 ± 2.3; p  =  0.023 vs. sham irradiation). PO-SCORAD values also decreased significantly in response to irradiation at 415 nm (-11.5 ± 18.4; p = 0.028 vs. sham irradiation). None of the other outcome measures (EASI, SCORAD, IGA, DLQI) changed significantly. No safety signals were observed. Evaluation of skin transcriptomes, cytokine levels in serum, and ELISpots from peripheral blood mononuclear cells isolated from a subset of patients revealed moderate decreases in IL-31 in response to irradiation with blue light. CONCLUSIONS: Despite its favorable safety profile and moderate reductions in itch and IL-31 levels, full-body blue light irradiation did not lead to an amelioration of any of the objective measures of AD.

Neurodegeneration or dysfunction in Phelan-McDermid syndrome? A multimodal approach with CSF and computational MRI.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare multisystem disease with global developmental delay and autistic features. Genetically, the disease is based on a heterozygous deletion of chromosome 22q13.3 with involvement of at least part of the SHANK3 gene or heterozygous pathogenic variants in SHANK3. Pathophysiologically, this syndrome has been regarded as a synaptopathy, but current data suggest an additional concept, since axonal functions of neurons are also impaired, thus, the specific pathophysiological processes in this disease are not yet fully understood. Since symptoms of the autism spectrum, regression, and stagnation in development occur, we investigated whether neuroinflammatory and neurodegenerative processes may also play a role. To this end, we analysed biomarkers in cerebrospinal fluid (CSF) and parameters from magnetic resonance imaging with high-resolution structural T1w volumetry and diffusion tensor imaging analysis in 19 Phelan-McDermid syndrome patients. RESULTS: CSF showed no inflammation but abnormalities in tau protein and amyloid-ß concentrations, however, with no typical biomarker pattern as in Alzheimer's disease. It could be demonstrated that these CSF changes were correlated with integrity losses of the fibres in the corticospinal tract as well as in the splenium and dorsal part of the cingulum. High CSF levels of tau protein were associated with loss of integrity of fibres in the corticospinal tract; lower levels of amyloid-ß were associated with decreasing integrity of fibre tracts of the splenium and posterior cingulate gyrus. Volumetric investigations showed global atrophy of the white matter, but not the grey matter, and particularly not in temporal or mesiotemporal regions, as is typical in later stages of Alzheimer's disease. CONCLUSIONS: In summary, alterations of neurodegenerative CSF markers in PMS individuals could be demonstrated which were correlated with structural connectivity losses of the corticospinal tract, the splenium, and the dorsal part of the cingulum, which can also be associated with typical clinical symptoms in these patients. These findings might represent a state of dysfunctional processes with ongoing degenerative and regenerative processes or a kind of accelerated aging. This study should foster further clinical diagnostics like tau- and amyloid-PET imaging as well as novel scientific approaches especially in basic research for further mechanistic proof.

Causal role of a promoter polymorphism in natural variation of the Arabidopsis floral repressor gene FLC.

Noncoding polymorphism frequently associates with phenotypic variation, but causation and mechanism are rarely established. Noncoding single-nucleotide polymorphisms (SNPs) characterize the major haplotypes of the Arabidopsis thaliana floral repressor gene FLOWERING LOCUS C (FLC). This noncoding polymorphism generates a range of FLC expression levels, determining the requirement for and the response to winter cold. The major adaptive determinant of these FLC haplotypes was shown to be the autumnal levels of FLC expression. Here, we investigate how noncoding SNPs influence FLC transcriptional output. We identify an upstream transcription start site (uTSS) cluster at FLC, whose usage is increased by an A variant at the promoter SNP-230. This variant is present in relatively few Arabidopsis accessions, with the majority containing G at this site. We demonstrate a causal role for the A variant at -230 in reduced FLC transcriptional output. The G variant upregulates FLC expression redundantly with the major transcriptional activator FRIGIDA (FRI). We demonstrate an additive interaction of SNP-230 with an intronic SNP+259, which also differentially influences uTSS usage. Combinatorial interactions between noncoding SNPs and transcriptional activators thus generate quantitative variation in FLC transcription that has facilitated the adaptation of Arabidopsis accessions to distinct climates.