Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Long-term monitoring of SARS-CoV-2 seroprevalence and variants in Ethiopia provides prediction for immunity and cross-immunity.

Under-reporting of COVID-19 and the limited information about circulating SARS-CoV-2 variants remain major challenges for many African countries. We analyzed SARS-CoV-2 infection dynamics in Addis Ababa and Jimma, Ethiopia, focusing on reinfection, immunity, and vaccination effects. We conducted an antibody serology study spanning August 2020 to July 2022 with five rounds of data collection across a population of 4723, sequenced PCR-test positive samples, used available test positivity rates, and constructed two mathematical models integrating this data. A multivariant model explores variant dynamics identifying wildtype, alpha, delta, and omicron BA.4/5 as key variants in the study population, and cross-immunity between variants, revealing risk reductions between 24% and 69%. An antibody-level model predicts slow decay leading to sustained high antibody levels. Retrospectively, increased early vaccination might have substantially reduced infections during the delta and omicron waves in the considered group of individuals, though further vaccination now seems less impactful.

Ancient mitogenomes from Pre-Pottery Neolithic Central Anatolia and the effects of a Late Neolithic bottleneck in sheep (Ovis aries).

Occupied between ~10,300 and 9300 years ago, the Pre-Pottery Neolithic site of Asikli Höyük in Central Anatolia went through early phases of sheep domestication. Analysis of 629 mitochondrial genomes from this and numerous sites in Anatolia, southwest Asia, Europe, and Africa produced a phylogenetic tree with excessive coalescences (nodes) around the Neolithic, a potential signature of a domestication bottleneck. This is consistent with archeological evidence of sheep management at Asikli Höyük which transitioned from residential stabling to open pasturing over a millennium of site occupation. However, unexpectedly, we detected high genetic diversity throughout Asikli Höyük's occupation rather than a bottleneck. Instead, we detected a tenfold demographic bottleneck later in the Neolithic, which caused the fixation of mitochondrial haplogroup B in southwestern Anatolia. The mitochondrial genetic makeup that emerged was carried from the core region of early Neolithic sheep management into Europe and dominates the matrilineal diversity of both its ancient and the billion-strong modern sheep populations.

A new etiology of nontraumatic C0-C1-C2-Complex instability - abnormality of musculus rectus capitis posterior minor: a case report.

PURPOSE: This study presents an abnormality of the musculus rectus capitis posterior minor (RCPmi) as a new etiological factor for nontraumatic sagittal plane instability in the C0-C1-C2-complex, with a focus on identifying the absence or atrophy of RCPmi on both sides. METHODS: A 36-year-old male patient presented with recurring neck pain (VAS 8/10) and tingling paresthesia in the entire left hand over a six-month period, without significant neurological deficits. Radiated arm pain was not reported. Imaging examinations revealed sagittal plane instability in the C0-C1-C2-complex, spinal canal stenosis (SCS), and myelopathy at the C1 level. Subsequently, a dorsal C0-1 reposition and fusion with laminectomy were performed. RESULTS: The congenital absence or atrophy of RCPmi, leading to the lack of cephalad-rearward traction on the C1-tuberculum-posterius, induced a developmental failure of the C1 posterior arch. Consequently, the oblate-shaped C1 posterior arch lost support from the underlying C2 posterior arch and the necessary cephalad-rearward traction throughout the patient's 36-year life. This gradual loss of support and traction caused the C1 posterior arch to shift gradually to the anterior side of the C2 posterior arch, resulting in a rotational subluxation centered on the C0/1 joints in the sagittal plane. Ultimately, this led to SCS and myelopathy. Traumatic factors were ruled out from birth to the present, and typical degenerative changes were not found in the upper cervical spine, neck muscles, and ligaments. CONCLUSION: In this case, we not only report the atrophy or absence of RCPmi as a new etiological factor for nontraumatic sagittal plane instability in the C0-C1-C2-complex but also discovered a new function of RCPmi. The cephalad-rearward traction exerted by RCPmi on the C1 posterior arch is essential for the development of a normal C1 anterior-posterior diameter.

TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

Impaired immune responses and prolonged viral replication in lung allograft recipients infected with SARS-CoV-2 in the early phase after transplantation.

PURPOSE: Lung transplant recipients are at increased risk of severe disease following infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) due to high-dose immunosuppressive drugs and the lung is the main organ affected by Coronavirus disease 2019 (COVID-19). Several studies have confirmed increased SARS-CoV-2-related mortality and morbidity in patients living with lung allografts; however, detailed immunological studies of patients with SARS-CoV-2 infection in the early phase following transplantation remain scarce. METHODS: We investigated patients who were infected with SARS-CoV-2 in the early phase (18-103 days) after receiving double-lung allografts (n = 4, LuTx) in comparison to immunocompetent patients who had not received solid organ transplants (n = 88, noTx). We analyzed SARS-CoV-2-specific antibody responses against the SARS-CoV-2 spike and nucleocapsid proteins using enzyme-linked immunosorbent assays (ELISA), chemiluminescence immunoassays (CLIA), and immunoblot assays. T cell responses were investigated using Elispot assays. RESULTS: One LuTx patient suffered from persistent infection with fatal outcome 122 days post-infection despite multiple interventions including remdesivir, convalescent plasma, and the monoclonal antibody bamlanivimab. Two patients experienced clinically mild disease with prolonged viral shedding (47 and 79 days), and one patient remained asymptomatic. Antibody and T cell responses were significantly reduced or undetectable in all LuTx patients compared to noTx patients. CONCLUSION: Patients in the early phase following lung allograft transplantation are vulnerable to infection with SARS-CoV-2 due to impaired immune responses. This patient population should be vaccinated before LuTx, protected from infection post-LuTx, and in case of infection treated generously with currently available interventions.

CSF3R T618I Collaborates With RUNX1-RUNX1T1 to Expand Hematopoietic Progenitors and Sensitizes to GLI Inhibition.

Activating colony-stimulating factor-3 receptor gene (CSF3R) mutations are recurrent in acute myeloid leukemia (AML) with t(8;21) translocation. However, the nature of oncogenic collaboration between alterations of CSF3R and the t(8;21) associated RUNX1-RUNX1T1 fusion remains unclear. In CD34+ hematopoietic stem and progenitor cells from healthy donors, double oncogene expression led to a clonal advantage, increased self-renewal potential, and blast-like morphology and distinct immunophenotype. Gene expression profiling revealed hedgehog signaling as a potential mechanism, with upregulation of GLI2 constituting a putative pharmacological target. Both primary hematopoietic cells and the t(8;21) positive AML cell line SKNO-1 showed increased sensitivity to the GLI inhibitor GANT61 when expressing CSF3R T618I. Our findings suggest that during leukemogenesis, the RUNX1-RUNXT1 fusion and CSF3R mutation act in a synergistic manner to alter hedgehog signaling, which can be exploited therapeutically.

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Ten rapid antigen tests for SARS-CoV-2 widely differ in their ability to detect Omicron-BA.4 and -BA.5.

Since late 2021, the variant landscape of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been dominated by the variant of concern (VoC) Omicron and its sublineages. We and others have shown that the detection of Omicron-BA.1 and -BA.2-positive respiratory specimens by rapid antigen tests (RATs) is impaired compared to Delta VoC-containing samples. Here, in a single-center retrospective laboratory study, we evaluated the performance of ten most commonly used RATs for the detection of Omicron-BA.4 and -BA.5 infections. We used 171 respiratory swab specimens from SARS-CoV-2 RNA-positive patients, of which 71 were classified as BA.4 and 100 as BA.5. All swabs were collected between July and September 2022. 50 SARS-CoV-2 PCR-negative samples from healthy individuals, collected in October 2022, showed high specificity in 9 out of 10 RATs. When assessing analytical sensitivity using clinical specimens, the 50% limit of detection (LoD50) ranged from 7.6 × 104 to 3.3 × 106 RNA copies subjected to the RATs for BA.4 compared to 6.8 × 104 to 3.0 × 106 for BA.5. Overall, intra-assay differences for the detection of these two Omicron subvariants were not significant for both respiratory swabs and tissue culture-expanded virus isolates. In contrast, marked heterogeneity was observed among the ten RATs: to be positive in these point-of-care tests, up to 443-fold (BA.4) and up to 56-fold (BA.5) higher viral loads were required for the worst performing RAT compared to the best performing RAT. True-positive rates for Omicron-BA.4- or -BA.5-containing specimens in the highest viral load category (Ct values < 25) ranged from 94.3 to 34.3%, dropping to 25.6 to 0% for samples with intermediate Ct values (25-30). We conclude that the high heterogeneity in the performance of commonly used RATs remains a challenge for the general public to obtain reliable results in the evolving Omicron subvariant-driven pandemic.

Automated antigen assays display a high heterogeneity for the detection of SARS-CoV-2 variants of concern, including several Omicron sublineages.

Diagnostic tests for direct pathogen detection have been instrumental to contain the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Automated, quantitative, laboratory-based nucleocapsid antigen (Ag) tests for SARS-CoV-2 have been launched alongside nucleic acid-based test systems and point-of-care (POC) lateral-flow Ag tests. Here, we evaluated four commercial Ag tests on automated platforms for the detection of different sublineages of the SARS-CoV-2 Omicron variant of concern (VoC) (B.1.1.529) in comparison with "non-Omicron" VoCs. A total of 203 Omicron PCR-positive respiratory swabs (53 BA.1, 48 BA.2, 23 BQ.1, 39 XBB.1.5 and 40 other subvariants) from the period February to March 2022 and from March 2023 were examined. In addition, tissue culture-expanded clinical isolates of Delta (B.1.617.2), Omicron-BA.1, -BF.7, -BN.1 and -BQ.1 were studied. These results were compared to previously reported data from 107 clinical "non-Omicron" samples from the end of the second pandemic wave (February to March 2021) as well as cell culture-derived samples of wildtype (wt) EU-1 (B.1.177), Alpha VoC (B.1.1.7) and Beta VoC (B.1.351)). All four commercial Ag tests were able to detect at least 90.9% of Omicron-containing samples with high viral loads (Ct < 25). The rates of true-positive test results for BA.1/BA.2-positive samples with intermediate viral loads (Ct 25-30) ranged between 6.7% and 100.0%, while they dropped to 0 to 15.4% for samples with low Ct values (> 30). This heterogeneity was reflected also by the tests' 50%-limit of detection (LoD50) values ranging from 44,444 to 1,866,900 Geq/ml. Respiratory samples containing Omicron-BQ.1/XBB.1.5 or other Omicron subvariants that emerged in 2023 were detected with enormous heterogeneity (0 to 100%) for the intermediate and low viral load ranges with LoD50 values between 23,019 and 1,152,048 Geq/ml. In contrast, detection of "non-Omicron" samples was more sensitive, scoring positive in 35 to 100% for the intermediate and 1.3 to 32.9% of cases for the low viral loads, respectively, corresponding to LoD50 values ranging from 6181 to 749,792 Geq/ml. All four assays detected cell culture-expanded VoCs Alpha, Beta, Delta and Omicron subvariants carrying up to six amino acid mutations in the nucleocapsid protein with sensitivities comparable to the non-VoC EU-1. Overall, automated quantitative SARS-CoV-2 Ag assays are not more sensitive than standard rapid antigen tests used in POC settings and show a high heterogeneity in performance for VoC recognition. The best of these automated Ag tests may have the potential to complement nucleic acid-based assays for SARS-CoV-2 diagnostics in settings not primarily focused on the protection of vulnerable groups. In light of the constant emergence of new Omicron subvariants and recombinants, most recently the XBB lineage, these tests' performance must be regularly re-evaluated, especially when new VoCs carry mutations in the nucleocapsid protein or immunological and clinical parameters change.

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.

IV Thrombolysis vs Early Dual Antiplatelet Therapy in Patients With Mild Noncardioembolic Ischemic Stroke.

BACKGROUND OBJECTIVES: It is unclear whether IV thrombolysis (IVT) outperforms early dual antiplatelet therapy (DAPT) in the acute setting of mild ischemic stroke. The aim of this study was to compare the early safety and efficacy of IVT with that of DAPT. METHODS: Data of mild noncardioembolic stroke patients with admission NIH Stroke Scale (NIHSS) score ≤3 who received IVT or early DAPT in the period 2018-2021 were extracted from a nationwide, prospective stroke unit registry. Study endpoints included symptomatic intracerebral hemorrhage (sICH), early neurologic deterioration ≥4 NIHSS points (END), and 3-month functional outcome by modified Rankin scale (mRS). RESULTS: A total of 1,195 mild stroke patients treated with IVT and 2,625 patients treated with DAPT were included. IVT patients were younger (68.1 vs 70.8 years), had less hypertension (72.8% vs 83.5%), diabetes (19% vs 28.8%), and a history of myocardial infarction (7.6% vs 9.2%), and slightly higher admission NIHSS scores (median 2 vs median 1) when compared with DAPT patients. After propensity score matching and multivariable adjustment, IVT was associated with sICH (4 [1.2%] vs 0) and END (adjusted odds ratio [aOR] 2.8, 95% CI 1.1-7.5), and there was no difference in mRS 0-1 at 3 months (aOR 1.3, 95% CI 0.7-2.6). DISCUSSION: This analysis from a prospective nationwide stroke unit network indicates that IVT is not superior to DAPT in the setting of mild noncardioembolic stroke and may eventually be associated with harm. Further research focusing on acute therapy of mild stroke is highly warranted. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that IVT is not superior to DAPT in patients with acute mild (NIHSS score ≤3) noncardioembolic stroke. The study lacks the statistical precision to exclude clinically important superiority of either therapy.

RNA polymerase II CTD is dispensable for transcription and required for termination in human cells.

The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.

German Ixodes inopinatus samples may not actually represent this tick species.

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) and whole genome sequencing of specific ticks (n = 8), we were able to determine that German samples, morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification, and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. Our results suggest that I. inopinatus is geographically isolated as a species (northern Africa and potentially southern Spain and Portugal) and brings into question whether I. inopinatus exists in central Europe. Our results highlight the probable existence of I. inopinatus and the power of utilizing genomic data in answering questions regarding tick taxonomy.

The genome of the reef-building glass sponge Aphrocallistes vastus provides insights into silica biomineralization.

Well-annotated and contiguous genomes are an indispensable resource for understanding the evolution, development, and metabolic capacities of organisms. Sponges, an ecologically important non-bilaterian group of primarily filter-feeding sessile aquatic organisms, are underrepresented with respect to available genomic resources. Here we provide a high-quality and well-annotated genome of Aphrocallistes vastus, a glass sponge (Porifera: Hexactinellida) that forms large reef structures off the coast of British Columbia (Canada). We show that its genome is approximately 80 Mb, small compared to most other metazoans, and contains nearly 2500 nested genes, more than other genomes. Hexactinellida is characterized by a unique skeletal architecture made of amorphous silicon dioxide (SiO2), and we identified 419 differentially expressed genes between the osculum, i.e. the vertical growth zone of the sponge, and the main body. Among the upregulated ones, mineralization-related genes such as glassin, as well as collagens and actins, dominate the expression profile during growth. Silicateins, suggested being involved in silica mineralization, especially in demosponges, were not found at all in the A. vastus genome and suggests that the underlying mechanisms of SiO2 deposition in the Silicea sensu stricto (Hexactinellida + Demospongiae) may not be homologous.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1.

The IFN system constitutes a powerful antiviral defense machinery. Consequently, effective IFN responses protect against severe COVID-19 and exogenous IFNs inhibit SARS-CoV-2 in vitro. However, emerging SARS-CoV-2 variants of concern (VOCs) may have evolved reduced IFN sensitivity. Here, we determined differences in replication and IFN susceptibility of an early SARS-CoV-2 isolate (NL-02-2020) and the Alpha, Beta, Gamma, Delta, and Omicron VOCs in Calu-3 cells, iPSC-derived alveolar type-II cells (iAT2) and air-liquid interface (ALI) cultures of primary human airway epithelial cells. Our data show that Alpha, Beta, and Gamma replicated to similar levels as NL-02-2020. In comparison, Delta consistently yielded higher viral RNA levels, whereas Omicron was attenuated. All viruses were inhibited by type-I, -II, and -III IFNs, albeit to varying extend. Overall, Alpha was slightly less sensitive to IFNs than NL-02-2020, whereas Beta, Gamma, and Delta remained fully sensitive. Strikingly, Omicron BA.1 was least restricted by exogenous IFNs in all cell models. Our results suggest that enhanced innate immune evasion rather than higher replication capacity contributed to the effective spread of Omicron BA.1.

Sequence dependent UV damage of complete pools of oligonucleotides.

Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments.

Quantitative trait locus for calving traits on Bos taurus autosome 18 in Holstein cattle is embedded in a complex genomic region.

Although the quantitative trait locus (QTL) on chromosome 18 (BTA18) associated with paternal calving ease and stillbirth in Holstein Friesian cattle and its cross has been known for over 20 years, to our knowledge, the exact causal genetic sequence has yet escaped identification. The aim of this study was to re-examine the region of the published QTL on BTA18 and to investigate the possible reasons behind this elusiveness. For this purpose, we carried out a combined linkage disequilibrium and linkage analysis using genotyping data of 2,697 German Holstein Friesian (HF) animals and subsequent whole-genome sequencing (WGS) data analyses and genome assembly of HF samples. We confirmed the known QTL in the 95% confidence interval of 1.089 Mbp between 58.34 and 59.43 Mbp on BTA18. Additionally, these 4 SNPs in the near-perfect linkage disequilibrium with the QTL haplotype were identified: rs381577268 (on 57,816,137 bp, C/T), rs381878735 (on 59,574,329 bp, A/T), rs464221818 (on 59,329,176 bp, C/T), and rs472502785 (on 59,345,689 bp, T/C). Search for the causal mutation using short and long-read sequences, and methylation data of the BTA18 QTL region did not reveal any candidates though. The assembly showed problems in the region, as well as an abundance of segmental duplications within and around the region. Taking the QTL of BTA18 in Holstein cattle as an example, the data presented in this study comprehensively characterize the genomic features that could also be relevant for other such elusive QTL in various other cattle breeds and livestock species as well.