Oxygen-Dependent Gene Expression in Development and Cancer: Lessons Learned from the Wilms’ Tumor Gene, WT1

Adequate tissue oxygenation is a prerequisite for normal development of the embryo. Most fetal organs are exquisitely susceptible to hypoxia which occurs when the delivery of oxygen is exceeded by the actual demand. Developmental abnormalities due to insufficient supply with oxygen can result from the impaired expression of genes with essential functions during embryogenesis. As such, the Wilms’ tumor gene, WT1, is among the fetal genes that are regulated by the local oxygen tension. WT1 was originally discovered as a tumor suppressor gene owing to loss-of-function mutations in a subset of pediatric renal neoplasias, known as nephroblastomas or Wilms’ tumors. Wilms’ tumors can arise when pluripotent progenitor cells in the embryonic kidney continue to proliferate rather than differentiating to glomeruli and tubules. WT1 encodes a zinc finger protein, of which multiple isoforms exist due to alternative mRNA splicing in addition to translational and post-translational modifications. While some WT1 isoforms function as transcription factors, other WT1 proteins are presumably involved in post-transcriptional mRNA processing. However, the role of WT1 reaches far beyond that of a tumor suppressor as homozygous disruption of Wt1 in mice caused embryonic lethality with a failure of normal development of the kidneys, gonads, heart, and other tissues. WT1 mutations in humans are associated with malformation of the genitourinary system. A common paradigm of WT1 expressing cells is their capacity to switch between a mesenchymal and epithelial state. Thus, WT1 likely acts as a master switch that enables cells to undergo reciprocal epithelial-to-mesenchymal transition. Impairment of renal precursor cells to differentiate along the epithelial lineage due to WT1 mutations may favor malignant tumor growth. This article shall provide a concise review of the function of WT1 in development and disease with special consideration of its regulation by molecular oxygen

On the Parametrization of Epidemiologic Models: Lessons from Modelling COVID-19 Epidemic

Numerous prediction models of SARS-CoV-2 pandemic were proposed in the past. Unknown parameters of these models are often estimated based on observational data. However, lag in case-reporting, changing testing policy or incompleteness of data lead to biased estimates. Moreover, parametrization is time-dependent due to changing age-structures, emerging virus variants, non-pharmaceutical interventions, and vaccination programs. To cover these aspects, we propose a principled approach to parametrize a SIR-type epidemiologic model by embedding it as a hidden layer into an input-output non-linear dynamical system (IO-NLDS). Observable data are coupled to hidden states of the model by appropriate data models considering possible biases of the data. This includes data issues such as known delays or biases in reporting. We estimate model parameters including their time-dependence by a Bayesian knowledge synthesis process considering parameter ranges derived from external studies as prior information. We applied this approach on a specific SIR-type model and data of Germany and Saxony demonstrating good prediction performances. Our approach can estimate and compare the relative effectiveness of non-pharmaceutical interventions and provide scenarios of the future course of the epidemic under specified conditions. It can be translated to other data sets, i.e., other countries and other SIR-type models

Enhanced pulmonary expression of the TrkB neurotrophin receptor in hypoxic rats is associated with increased acetylcholine-induced airway contractility

AIM We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1alpha. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. METHODS Rats were exposed either to 21% O(2) or 8% O(2) for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. RESULTS TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l-NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. CONCLUSION These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO

Brucella microti: the genome sequence of an emerging pathogen

<p>Abstract</p> <p>Background</p> <p>Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel <it>Brucella </it>species, <it>Brucella microti</it>, was determined. <it>B. microti </it>is a member of the genus <it>Brucella </it>within the <it>Alphaproteobacteria</it>, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other <it>Brucella </it>species, <it>B. microti </it>is a fast growing and biochemically very active microorganism with a phenotype more similar to that of <it>Ochrobactrum</it>, a facultative human pathogen. The atypical phenotype of <it>B. microti </it>prompted us to look for genomic differences compared to other <it>Brucella </it>species and to look for similarities with <it>Ochrobactrum</it>.</p> <p>Results</p> <p>The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of <it>B. microti </it>is almost identical to that of <it>Brucella suis </it>1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in <it>B. microti</it>. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between <it>B. microti </it>and <it>Ochrobactrum anthropi </it>but impaired in other sequenced <it>Brucella</it>. The most noticeable difference between <it>B. microti </it>and other <it>Brucella </it>species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of <it>B. microti</it>.</p> <p>Conclusion</p> <p>Contrary to expectations from the phenotypic analysis, the genome sequence of <it>B. microti </it>is highly similar to that of known <it>Brucella </it>species, and is remotely related to the one of <it>O. anthropi</it>. How the few differences in gene content between <it>B. microti </it>and <it>B. suis </it>1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the <it>Brucella </it>genus. The genome sequence of <it>B. microti </it>will serve as a model for differential expression analysis and complementation studies. Our results also raise some concerns about the importance given to phenotypical traits in the definition of bacterial species.</p

Ulceroglandular form of tularemia after squirrel bite: a case report

Background The diagnosis of tularemia is not often considered in Germany as the disease is still rare in this country. Nonetheless, Francisella tularensis, the causative agent of tularemia, can infect numerous animal species and should, therefore, not be neglected as a dangerous pathogen. Tularemia can lead to massively swollen lymph nodes and might even be fatal without antibiotic treatment. To our knowledge, the case described here is the first report of the disease caused by a squirrel bite in Germany. Case presentation A 59-year-old German woman with a past medical history of hypothyroidism and cutaneous lupus erythematosus presented at the emergency room at St. Katharinen Hospital with ongoing symptoms and a swollen right elbow persisting despite antibiotic therapy with cefuroxime for 7 days after she had been bitten (right hand) by a wild squirrel (Eurasian red squirrel). After another 7 days of therapy with piperacillin/tazobactam, laboratory analysis using real-time polymerase chain reaction (PCR) confirmed the suspected diagnosis of tularemia on day 14. After starting the recommended antibiotic treatment with ciprofloxacin, the patient recovered rapidly. Conclusion This is the first report of a case of tularemia caused by a squirrel bite in Germany. A naturally infected squirrel has recently been reported in Switzerland for the first time. The number of human cases of tularemia has been increasing over the last years and, therefore, tularemia should be taken into consideration as a diagnosis, especially in a patient bitten by an animal who also presents with headache, increasing pain, lymphadenitis, and fever, as well as impaired wound healing. The pathogen can easily be identified by a specific real-time PCR assay of wound swabs and/or by antibody detection, for example by enzyme-linked immunosorbent assay (ELISA), if the incident dates back longer than 2 weeks.Peer Reviewe

A one-step PCR assay for the detection of pathogenic Y. enterocolitica in artificially contaminated fecal samples and lymphoid tissue

In order to specifically detect pathogenic, plasmid bearing Yersinia enterocolitica, we have developed a polymerase chain reaction (PCR) assay based on the plasmid located gene yopT. A substantial number of mismatches within the yopT coding sequence between Y. enterocolitca, Y. pseudotuberculosis, and Y. pestis was used to generate a primer pair that exclusively detects pathogenic Y. enteroco/itica with a high sensitivity and specificity. When this PCR assay was used for the detection of pathogenic Y. enterocolilica cells in artificially inoculated fecal samples and lymphoid tissue of pigs, levels as low as 102 cells per gram feces and 101 cells per gram lymphoid tissue could be detected if an 24 h pre-enrichment in Luria Bertani-Bouillon was performed prior to the PCR

Phorbol-Ester Mediated Suppression of hASH1 Synthesis: Multiple Ways to Keep the Level Down

Human achaete-scute homolog-1 (hASH1), encoded by the human ASCL1 gene, belongs to the family of basic helix-loop-helix transcription factors. hASH1 and its mammalian homolog Mash1 are expressed in the central and peripheral nervous system during development, and promote early neuronal differentiation. Furthermore, hASH1 is involved in the specification of neuronal subtype identities. Misexpression of the transcription factor is correlated with a variety of tumors, including lung cancer and neuroendocrine tumors. To gain insights into the molecular mechanisms of hASH1 regulation, we screened for conditions causing changes in hASH1 gene expression rate. We found that treatment of human neuroblastoma-derived Kelly cells with phorbol 12-myristate 13-acetate (PMA) resulted in a fast, strong and long-lasting suppression of hASH1 synthesis. Reporter gene assays with constructs, in which the luciferase activity was controlled either by the ASCL1 promoter or by the hASH1 mRNA untranslated regions (UTRs), revealed a mainly UTR-dependent mechanism. The hASH1 promoter activity was decreased only after 48 h of PMA administration. Our data indicate that different mechanisms acting consecutively at the transcriptional and post-transcriptional level are responsible for hASH1 suppression after PMA treatment. We provide evidence that short term inhibition of hASH1 synthesis is attributed to hASH1 mRNA destabilization, which seems to depend mainly on protein kinase C activity. Under prolonged conditions (48 h), hASH1 suppression is mediated by decreased promoter activity and inhibition of mRNA translation

Maize Source Leaf Adaptation to Nitrogen Deficiency Affects Not Only Nitrogen and Carbon Metabolism But Also Control of Phosphate Homeostasis

Schlueter U, Mascher M, Colmsee C, et al. Maize Source Leaf Adaptation to Nitrogen Deficiency Affects Not Only Nitrogen and Carbon Metabolism But Also Control of Phosphate Homeostasis. Plant Physiology. 2012;160(3):1384-1406.Crop plant development is strongly dependent on the availability of nitrogen (N) in the soil and the efficiency of N utilization for biomass production and yield. However, knowledge about molecular responses to N deprivation derives mainly from the study of model species. In this article, the metabolic adaptation of source leaves to low N was analyzed in maize (Zea mays) seedlings by parallel measurements of transcriptome and metabolome profiling. Inbred lines A188 and B73 were cultivated under sufficient (15 mM) or limiting (0.15 mM) nitrate supply for up to 30 d. Limited availability of N caused strong shifts in the metabolite profile of leaves. The transcriptome was less affected by the N stress but showed strong genotype-and age-dependent patterns. N starvation initiated the selective down-regulation of processes involved in nitrate reduction and amino acid assimilation; ammonium assimilation-related transcripts, on the other hand, were not influenced. Carbon assimilation-related transcripts were characterized by high transcriptional coordination and general down-regulation under low-N conditions. N deprivation caused a slight accumulation of starch but also directed increased amounts of carbohydrates into the cell wall and secondary metabolites. The decrease in N availability also resulted in accumulation of phosphate and strong down-regulation of genes usually involved in phosphate starvation response, underlining the great importance of phosphate homeostasis control under stress conditions

Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein–Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail

Comparison of scoring methods for the detection of causal genes with or without rare variants

Rare causal variants are believed to significantly contribute to the genetic basis of common diseases or quantitative traits. Appropriate statistical methods are required to discover the highest possible number of disease-relevant variants in a genome-wide screening study. The publicly available Genetic Analysis Workshop 17 data set consists of 697 individuals and 24,487 genetic variants. It includes a simulated complex disease model with intermediate quantitative phenotypes. We compare four gene-wise scoring methods with respect to ranking of causal genes under variable allele frequency thresholds for collapsing of rare variants and considering whether or not rare variants were included. We also compare causal genes for which the ranks differ clearly between scoring methods regarding such characteristics as number and strength of causal variants. We corroborated our findings with additional simulations. We found that the maximum statistics method was superior in assigning high ranks to genes with a single strong causal variant. Hotelling’s T2 test was superior for genes with several independent causal variants. This was consistent for all phenotypes and was confirmed by single-gene analyses and additional simulations. The multivariate analysis performed similarly to Hotelling’s T2 test. The least absolute shrinkage and selection operator (LASSO) analysis was widely comparable with the maximum statistics method. We conclude that the maximum statistics method is a superior alternative to Hotelling’s T2 test if one expects only one independent causal variant per gene with a dominating effect. Such a variant could also be a supermarker derived by collapsing rare variants. Because the true nature of the genetic effect is unknown for real data, both methods need to be taken into consideration