Functional interactions of the Transcription Factor B during transcription initiation in Pyrococcus furiosus

The preinitiation complex of the transcription machinery in archaeal organisms resembles a simplified version of the eukaryotic RNA polymerase II transcription system. Both systems share homologous general transcription factors to recruit RNA polymerase to the promoter to initiate RNA synthesis. The transcription factor (II)B plays an important role during transcription initiation. Based on eukaryotic cryo-EM and crystal structures several functional interactions and structural transitions of TF(II)B were proposed. To detect specific interactions of the archaeal P. furiosus TFB during transcription initiation different in vitro transcription assays were performed. In addition, the replication protein A of P. furiosus was also investigated using various in vitro experiments. Crosslinking experiments using TFB, which contained a UV inducible photo crosslinker, and site-specific radioactively labeled DNA templates revealed an almost similar topology of the archaeal TFB B-reader and B-linker domains in the preinitiation complex in comparison to corresponding regions predicted in eukaryotic structures. Unlike it was postulated in open complex models, the non-transcribed strand is located closer to the B-linker strand than the B-linker helix. The B-core amino acid F192 contact DNA 19 nucleotides upstream the transcribed strand, in accordance to a published crystal of P. woesei TATA/TBP/TFB-core structure, but is different to predicted eukaryotic closed and open complex models. Crosslinking experiments in stalled complexes showed that RNA interacts with the B-reader loop at a length of 6nt, and further clashes with the B-reader helix domain with a length of 8nt. At register +10 the TFB B-reader is displaced, which causes collapse of the transcription bubble. It was also demonstrated that TFB is present at register +6 to +14 in the complex, and tended to be released from register +15 onwards, indicating a destabilization of TFB at register +13/+14. Alanine substitutions of amino acids of the TFB B-reader loop revealed that this region mainly stabilizes the transcription bubble due to charge-dependent interactions with the transcribing strand. In contrast to the predicted RNA-DNA separation model derived from a eukaryotic initially transcribing complex, RNA-strand separation does not depend on the charge of the PfuTFB B-reader loop. Single molecule FRET experiments revealed that DNA bending depends on the presence of TFB in P. furiosus. In vitro transcription assays with RPA showed that this protein has binding preference to single stranded DNA. Experiments further showed that RPA is not involved in transcription initiation, but it stimulates transcription. Therefore RPA functions during elongation of transcription, possibly due to a stabilization of the RNA polymerase and increase of the processivity. The results presented here give a more detailed insight into molecular interactions of TFB and are the first biochemical data on dynamic rearrangements of TFB during transcription initiation and transition to early elongation. It further deepens the understanding of archaeal transcription processes and complements structural information derived from related eukaryotic organisms

Displacement of the transcription factor B reader domain during transcription initiation

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards

Mitosis domain generalization in histopathology images -- The MIDOG challenge

The density of mitotic figures within tumor tissue is known to be highly correlated with tumor proliferation and thus is an important marker in tumor grading. Recognition of mitotic figures by pathologists is known to be subject to a strong inter-rater bias, which limits the prognostic value. State-of-the-art deep learning methods can support the expert in this assessment but are known to strongly deteriorate when applied in a different clinical environment than was used for training. One decisive component in the underlying domain shift has been identified as the variability caused by using different whole slide scanners. The goal of the MICCAI MIDOG 2021 challenge has been to propose and evaluate methods that counter this domain shift and derive scanner-agnostic mitosis detection algorithms. The challenge used a training set of 200 cases, split across four scanning systems. As a test set, an additional 100 cases split across four scanning systems, including two previously unseen scanners, were given. The best approaches performed on an expert level, with the winning algorithm yielding an F_1 score of 0.748 (CI95: 0.704-0.781). In this paper, we evaluate and compare the approaches that were submitted to the challenge and identify methodological factors contributing to better performance.Comment: 19 pages, 9 figures, summary paper of the 2021 MICCAI MIDOG challeng

A review of the surgical options for the correction of presbyopia

Presbyopia is an age-related eye condition where one of the signs is the reduction in the amplitude of accommodation, resulting in the loss of ability to change the eye's focus from far to near. It is the most common age-related ailments affecting everyone around their mid-40s. Methods for the correction of presbyopia include contact lens and spectacle options but the surgical correction of presbyopia still remains a significant challenge for refractive surgeons. Surgical strategies for dealing with presbyopia may be extraocular (corneal or scleral) or intraocular (removal and replacement of the crystalline lens or some type of treatment on the crystalline lens itself). There are however a number of limitations and considerations that have limited the widespread acceptance of surgical correction of presbyopia. Each surgical strategy presents its own unique set of advantages and disadvantages. For example, lens removal and replacement with an intraocular lens may not be preferable in a young patient with presbyopia without a refractive error. Similarly treatment on the crystalline lens may not be a suitable choice for a patient with early signs of cataract. This article is a review of the options available and those that are in development stages and are likely to be available in the near future for the surgical correction of presbyopia

Presbyopia:Effectiveness of correction strategies

Presbyopia is a global problem affecting over a billion people worldwide. The prevalence of unmanaged presbyopia is as high as 50% of those over 50 years of age in developing world populations due to a lack of awareness and accessibility to affordable treatment, and is even as high as 34% in developed countries. Definitions of presbyopia are inconsistent and varied, so we propose a redefinition that states “presbyopia occurs when the physiologically normal age-related reduction in the eye's focusing range reaches a point, when optimally corrected for distance vision, that the clarity of vision at near is insufficient to satisfy an individual's requirements”. Presbyopia is inevitable if one lives long enough, but intrinsic and extrinsic risk factors including cigarette smoking, pregnancy history, hyperopic or astigmatic refractive error, ultraviolet radiation, female sex (although accommodation is similar to males), hotter climates and some medical conditions such as diabetes can accelerate the onset of presbyopic symptoms. Whilst clinicians can ameliorate the symptoms of presbyopia with near vision spectacle correction, bifocal and progressive spectacle lenses, monovision, translating or multifocal contact lenses, monovision, extended depth of focus, multifocal (refractive, diffractive and asymmetric designs) or ‘accommodating’ intraocular lenses, corneal inlays, scleral expansion, laser refractive surgery (corneal monovision, corneal shrinkage, corneal multifocal profiles and lenticular softening), pharmacologic agents, and electro-stimulation of the ciliary muscle, none fully overcome presbyopia in all patients. While the restoration of natural accommodation or an equivalent remains elusive, guidance is gives on presbyopic correction evaluation techniques