The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats--CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. A hallmark of CRISPR-Cas is the ...involvement of short crRNAs that guide associated proteins in the destruction of invading DNA or RNA. We present three fundamentally distinct processing pathways in the cyanobacterium Synechocystis sp. PCC6803 for a subtype I-D (CRISPR1), and two type III systems (CRISPR2 and CRISPR3), which are located together on the plasmid pSYSA. Using high-throughput transcriptome analyses and assays of transcript accumulation we found all CRISPR loci to be highly expressed, but the individual crRNAs had profoundly varying abundances despite single transcription start sites for each array. In a computational analysis, CRISPR3 spacers with stable secondary structures displayed a greater ratio of degradation products. These structures might interfere with the loading of the crRNAs into RNP complexes, explaining the varying abundancies. The maturation of CRISPR1 and CRISPR2 transcripts depends on at least two different Cas6 proteins. Mutation of gene sll7090, encoding a Cmr2 protein led to the disappearance of all CRISPR3-derived crRNAs, providing in vivo evidence for a function of Cmr2 in the maturation, regulation of expression, Cmr complex formation or stabilization of CRISPR3 transcripts. Finally, we optimized CRISPR repeat structure prediction and the results indicate that the spacer context can influence individual repeat structures.
An RNA-based screen was performed to reveal a possible evolutionary scenario for the CRISPR-Cas systems in two cyanobacterial model strains. Following the analysis of a draft genome sequence of ...Synechocystis sp PCC6714, three different CRISPR-Cas systems were characterized that have different degrees of relatedness to another three CRISPR-Cas systems in Synechocystis sp PCC6803. A subtype III-B system was identified that is extremely conserved between both strains. Strong signals in northern hybridizations and the presence of different spacers (but identical repeats) indicated this system to be active, despite the absence of a known endonuclease candidate gene involved in the maturation of its crRNAs in the two strains. The other two systems were found to differ significantly from each other, with different sets of repeat-spacer arrays and different Cas genes. In view of the otherwise very close relatedness of the two analyzed strains, this is suggestive of an unknown mechanism involved in the replacement of CRISPR-Cas cassettes as a whole. Further RNA analyses revealed the accumulation of crRNAs to be impacted by environmental conditions critical for photoautotropic growth. All six systems are associated with a gene for a possible transcriptional repressor. Indeed, we identified one of these genes, sll7009, as encoding a negative regulator specific for the CRISPR1 subtype I-D system in Synechocystis sp PCC6803.
A hallmark of defense mechanisms based on clustered regularly interspaced short palindromic repeats (CRISPR) and associated sequences (Cas) are the crRNAs that guide these complexes in the ...destruction of invading DNA or RNA. Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Based on genetic and transcriptomic evidence, two associated endoribonucleases, Cas6-1 and Cas6-2a, were postulated to be involved in crRNA maturation from CRISPR1 or CRISPR2, respectively. Here, we report a promiscuity of both enzymes to process in vitro not only their cognate transcripts, but also the respective non-cognate precursors, whereas they are specific in vivo Moreover, while most of the repeats serving as substrates were cleaved in vitro, some were not. RNA structure predictions suggested that the context sequence surrounding a repeat can interfere with its stable folding. Indeed, structure accuracy calculations of the hairpin motifs within the repeat sequences explained the majority of analyzed cleavage reactions, making this a good measure for predicting successful cleavage events. We conclude that the cleavage of CRISPR1 and CRISPR2 repeat instances requires a stable formation of the characteristic hairpin motif, which is similar between the two types of repeats. The influence of surrounding sequences might partially explain variations in crRNA abundances and should be considered when designing artificial CRISPR arrays.
Erythema multiforme (EM), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) comprise a family of mucocutaneous diseases associated with significant morbidity and mortality. ...Previous studies have confirmed the presence of autoantibodies to desmoplakin (Dp) I and II in patients with EM, SJS, and TEN. Truncated Dp production leads to characteristic changes visible on light microscopy: perinuclear clumping of keratin filaments and dyskeratotic keratinocyte. Based on these observations, the question arises as to whether a loss of Dp immunoreactivity in skin biopsies could serve as a diagnostic marker of EM, SJS, and TEN. This study analyzed Dp immunostaining patterns in 20 patients with EM or SJS/TEN. To assess the specificity of this approach, Dp immunostaining was also performed on specimens from patients with 5 potential histologic mimics of EM, SJS, and TEN. All of the samples from patients with EM, SJS, and TEN demonstrated absent or markedly diminished staining for Dp. A χ test demonstrated a statistically significant difference between the staining patterns in EM, SJS, and TEN and each of the other diagnostic groups that were investigated. This is the first report demonstrating statistically significant specificity of Dp staining patterns in EM/SJS/TEN as compared with other interface dermatitides.
Aerosol generation, deposition and transport in the PADDOCC system.
Absorption studies with aerosol formulation delivered by metered dose inhalers across cell- and tissue-based
in vitro models of the ...pulmonary epithelia are not trivial due to the complexity of the processes involved: (i) aerosol generation and deposition, (ii) drug release from the carrier, and (iii) absorption across the epithelial air–blood barrier. In contrast to the intestinal mucosa, pulmonary epithelia are only covered by a thin film of lining fluid. Submersed cell culture systems would not allow to studying the deposition of aerosol particles and their effects on this delicate epithelial tissue.
We developed a new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to mimic the inhalation of a single metered aerosol dose and its subsequent deposition on filter-grown pulmonary epithelial cell monolayers exposed to an air–liquid interface. The reproducibility of deposition of these dry powder aerosols and subsequent drug transport across Calu-3 monolayers with commercially available dry powder inhalers containing salbutamol sulphate or budesonide could be demonstrated.
In the context of developing new dry powder aerosol formulations, PADDOCC appears as a useful tool, allowing reducing animal testing and faster translation into clinical trials.
Description of Referrals for Colposcopy in a Hospital in Brazil de Carvalho, Stephanie Hein; Rosa, Dayana Aparecida Nacimento; Santos, André Luis Ferreira ...
Revista Brasileira de ginecologia e obstetrícia,
03/2020, Letnik:
42, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Abstract
Objective
To describe the referral for colposcopy in a Hospital in Brazil and the relative frequency of patients who benefited from it, considering the correct indications for the ...examination and its final diagnoses.
Methods
A retrospective study was performed in the colposcopy service database of the Hospital Universitário de Taubaté, Taubaté, state of São Paulo, Brazil. The frequency validated in the analysis of the medical records of women referred for clinical indication or cytological alteration, attended from March 2015 to March 2017. The population selected and analyzed included 256 results that were correlated to the cytological, clinical data and the result of the colposcopy.
Results
Of the women referred, 45% presented out of the age of screening according to the guidelines of cervical cancer screening, 8.6% being adolescents and young adults < 25 years old, and 36.4% of the patients being ≥ 65 years old. A total of 50% of the patients had no indication of colposcopy, that is, normal cytologies, benign changes, ectopia, cervicitis, atypical squamous cells of indeterminate significance (ASC-US) and low-grade intraepithelial lesion (LSIL) without persistence and normal clinical appearance. A total of 39.84% who underwent colposcopy had high-grade lesion or cancer results, thus benefiting from the adequate referral.
Conclusion
Most (60.16%) of the patients referred to the colposcopy service did not benefit from the referral for results without changes, such as negative colposcopies, histologies with no cervical intraepithelial neoplasm (CIN) or only CIN 1, or were out of the age for screening. These findings therefore demonstrate a significant number of unnecessary and inadequate referrals.
The blood-air barrier formed by the alveolar epithelium of the peripheral lung is crucial for the pulmonary delivery of drugs. Most existing in vitro models mimicking the blood-air barrier are ...represented by tumor cells or immortalized cells and lack biological relevance due to their genetic alterations and underexpressed essential physiological functions. However, the increasing interest of aerosol administration of medicines to the respiratory system requires the development and use of representative in vitro models. Thereby, human alveolar epithelial cells (hAEpC) are a suitable test system allowing standardized toxicity and transport studies for newly developed compounds and delivery systems. The isolation, purification, and cultivation of hAEpC are described as well as their possible application in the so-called Pharmaceutical Aerosol Deposition Device On Cell Cultures (PADDOCC) mimicking the complete inhalation process of a powder aerosol in vitro.
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