SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

Transcription readthrough is prevalent in healthy human tissues and associated with inherent genomic features.

Transcription termination is a crucial step in the production of conforming mRNAs and functional proteins. Under cellular stress conditions, the transcription machinery fails to identify the termination site and continues transcribing beyond gene boundaries, a phenomenon designated as transcription readthrough. However, the prevalence and impact of this phenomenon in healthy human tissues remain unexplored. Here, we assessed transcription readthrough in almost 3000 transcriptome profiles representing 23 human tissues and found that 34% of the expressed protein-coding genes produced readthrough transcripts. The production of readthrough transcripts was restricted in genomic regions with high transcriptional activity and was associated with inefficient splicing and increased chromatin accessibility in terminal regions. In addition, we showed that these transcripts contained several binding sites for the same miRNA, unravelling a potential role as miRNA sponges. Overall, this work provides evidence that transcription readthrough is pervasive and non-stochastic, not only in abnormal conditions but also in healthy tissues. This suggests a potential role for such transcripts in modulating normal cellular functions.

Thulium laser vaporesection of prostates with volume exceeding 100 cm3 as an alternative to HoLEP and ThuLEP.

The aim of this study is to evaluate the outcomes of thulium laser vaporesection of prostates with volume exceeding 100 cm3. In the present prospective study, patients with infra-vesical urinary obstruction due to a prostate with volume exceeding 100 cm3 underwent endoscopic vaporesection using thulium laser. In this procedure, prostate chips were resected without morcellation. The technical aspects of surgery, admission time, post-operative catheter time and post-operative complications were analyzed. Flowmetry was performed combined with prostatic ultrasound in the follow-up. Between March 2010 and November 2018, 156 cases with benign prostatic hyperplasia (BPH; volume >100 cm3) were treated. The mean patient age was 67.8 years (48.4-86.6 years), and the mean prostatic volume was 137 cm3 (100-436 cm3). The mean length of hospitalization was 1.48 days (1-8 days), and the mean post-operative catheter time was 5.1 (1-17). Three cases (1.9%) required readmission due to hematuria. The mean follow-up time was 31.2 months (standard deviation = 27.7). Urethral stricture was observed in 14 cases (9%), with bulbar urethra being the most frequent finding. Urinary tract infection was observed in 11 cases (7.1%), and urinary incontinence was observed in 5 cases. The mean peak urinary flow at 12 and 24 months was 26.9 ± 12.5 and 23.9 ± 11.7 ml/s, respectively, and the mean urinary flow during the final follow-up at 41 months was 21.6 ml/s. Thulium laser vaporesection is a valid alternative to open prostatectomy, HoLEP and ThuLEP in patients with large BPH. Urinary flow remained elevated throughout the follow-up.

Patient Satisfaction with Healthcare Services and the Techniques Used for its Assessment: A Systematic Literature Review and a Bibliometric Analysis.

Patient satisfaction with healthcare provision services and the factors influencing it are be-coming the main focus of many scientific studies. Assuring the quality of the provided services is essential for the fulfillment of patients' expectations and needs. Thus, this systematic review seeks to find the determinants of patient satisfaction in a global setting. We perform an analysis to evaluate the collected literature and to fulfill the literature gap of bibliometric analysis within this theme. This review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) approach. We conducted our database search in Scopus, Web of Science, and PubMed in June 2022. Studies from 2000-2021 that followed the inclusion and exclusion criteria and that were written in English were included in the sample. We ended up with 157 articles to review. A co-citation and bibliographic coupling analysis were employed to find the most relevant sources, authors, and documents. We divided the factors influencing patient satisfaction into criteria and explanatory variables. Medical care, communication with the patient, and patient's age are among the most critical factors for researchers. The bibliometric analysis revealed the countries, institutions, documents, authors, and sources most productive and significant in patient satisfaction.

High Sensitivity Cryogenic Temperature Sensors Based on Arc-Induced Long-Period Fiber Gratings.

In this paper, we investigated the evolution of the dispersion curves of long-period fiber gratings (LPFGs) from room temperature down to 0 K. We considered gratings arc-induced in the SMF28 fiber and in two B/Ge co-doped fibers. Computer simulations were performed based on previously published experimental data. We found that the dispersion curves belonging to the lowest-order cladding modes are the most affected by the temperature changes, but those changes are minute when considering cladding modes with dispersion turning points (DTP) in the telecommunication windows. The temperature sensitivity is higher for gratings inscribed in the B/Ge co-doped fibers near DTP and the optimum grating period can be chosen at room temperature. A temperature sensitivity as high as -850 pm/K can be obtained in the 100-200 K temperature range, while a value of -170 pm/K is reachable at 20 K.

Optical Strain Gauge Prototype Based on a High Sensitivity Balloon-like Interferometer and Additive Manufacturing.

An optical strain gauge based on a balloon-like interferometer structure formed by a bent standard single-mode fiber combined with a 3D printer piece has been presented and demonstrated, which can be used to measure displacement. The interferometer has a simple and compact size, easy fabrication, low cost, and is repeatable. The sensor is based on the interference between the core and cladding modes. This is caused by the fiber's curvature because when light propagates through the curved balloon-shaped interferometer region, a portion of it will be released from the core limitation and coupled to the cladding. The balloon has an axial displacement as a result of how the artwork was constructed. The sensor head is sandwiched between two cantilevers such that when there is a displacement, the dimension associated with the micro bend is altered. The sensor response as a function of displacement can be determined using wavelength shift or intensity change interrogation techniques. Therefore, this optical strain gauge is a good option for applications where structure displacement needs to be examined. The sensor presents a sensitivity of 55.014 nm for displacement measurements ranging from 0 to 10 mm and a strain sensitivity of 500.13 pm/µÏµ.

Aerocyte specification and lung adaptation to breathing is dependent on alternative splicing changes.

Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found that <i>Vegfa</i> switches from the <i>Vegfa</i> 164 isoform to the longer <i>Vegfa</i> 188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type-specific regulation of <i>Vegfa</i> alternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.

A Simple Optical Sensor Based on Multimodal Interference Superimposed on Additive Manufacturing for Diameter Measurement.

In many areas, the analysis of a cylindrical structure is necessary, and a form to analyze it is by evaluating the diameter changes. Some areas can be cited: pipelines for oil or gas distribution and radial growth of trees whose diameter changes are directly related to irrigation and the radial expansion since it depends on the water soil deficit. For some species, these radial variations can change in around 5 mm. This paper proposes and experimentally investigates a sensor based on a core diameter mismatch technique for diameter changes measurement. The sensor structure is a combination of a cylindrical piece developed using a 3D printer and a Mach-Zehnder interferometer. The pieces were developed to assist in monitoring the diameter variation. It is formed by splicing an uncoated short section of MMF (Multimode Fiber) between two standard SMFs (Singlemode Fibers) called SMF-MMF-SMF (SMS), where the MMF length is 15 mm. The work is divided into two main parts. Firstly, the sensor was fixed at two points on the first developed piece, and the diameter reduction caused dips or peaks shift of the transmittance spectrum due to curvature and strain influence. The fixation point (FP) distances used are: 5 mm, 10 mm, and 15 mm. Finally, the setup with the best sensitivity was chosen, from first results, to develop another test with an optimization. This optimization is performed in the printed piece where two supports are created so that only the strain influences the sensor. The results showed good sensitivity, reasonable dynamic range, and easy setup reproduction. Therefore, the sensor could be used for diameter variation measurement for proposed applications.

In vitro reconstitution of Escherichia coli divisome activation.

The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops.

DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.

Simulation of the Transmission Spectrum of Long-Period Fiber Gratings Structures with a Propagating Acoustic Shock Front.

In this paper, we investigate modification of transmission spectra of long-period fiber grating structures with an acoustic shock front propagating along the fiber. We simulate transmission through inhomogeneous long-period fiber gratings, π-shift and reflective π-shift gratings deformed by an acoustic shock front. Coupled mode equations describing interaction of co-propagating modes in a long-period fiber grating structures with inhomogeneous deformation are used for the simulation. Two types of apodization are considered for the grating modulation amplitude, such as uniform and raised-cosine. We demonstrate how the transmission spectrum is produced by interference between the core and cladding modes coupled at several parts of the gratings having different periods. For the π-shift long-period fiber grating having split spectral notch, the gap between the two dips becomes several times wider in the grating with the acoustic wave front than the gap in the unstrained grating. The behavior of reflective long-period fiber gratings depends on the magnitude of the phase shift near the reflective surface: an additional dip is formed in the 0-shift grating and the short-wavelength dip disappears in the π-shift grating.

Optical Fiber Interferometers Based on Arc-Induced Long Period Gratings at INESC TEC.

In this work, we review the most important achievements of an INESC TEC long-period-grating-based fiber optic Michelson and Mach-Zehnder configuration modal interferometer with coherence addressing and heterodyne interrogation as a sensing structure for measuring environmental refractive index and temperature. The theory for Long Period Grating (LPG) interferometers and coherence addressing and heterodyne interrogation is presented. To increase the sensitivity to external refractive index and temperature, several LPG interferometers parameters are studied, including order of cladding mode, a reduction of the fiber diameter, different type of fiber, cavity length and the antisymmetric nature of cladding modes.

Arc-Induced Long-Period Fiber Gratings at INESC TEC. Part II: Properties and Applications in Optical Communications and Sensing.

In this work, we review the most important achievements of INESC TEC related to the properties and applications of arc-induced long-period fiber gratings. The polarization dependence loss, the spectral behavior at temperatures ranging from cryogenic up to 1200 °C and under exposure to ultraviolet and gamma radiation is described. The dependence of gratings sensitivity on the fabrication parameters is discussed. Several applications in optical communications and sensing domains are referred.

Arc-Induced Long-Period Fiber Gratings at INESC TEC. Part I: Fabrication, Characterization and Mechanisms of Formation.

In this work, we reviewed the most important achievements of INESC TEC related to the fabrication of long-period fiber gratings using the electric arc technique. We focused on the fabrication setup, the type of fiber used, and the effect of the fabrication parameters on the gratings' transmission spectra. The theory was presented, as well as a discussion on the mechanisms responsible for the formation of the gratings, supported by the measurement of the temperature reached by the fiber during an electric arc discharge.

Phenotypic and Genotypic Drug Susceptibility Assessment of Mycobacterium bovis Bacillus Calmette-Guérin Clinical Strains.

PURPOSE: Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine licensed against tuberculosis. Despite the protection offered by the vaccine, in some circumstances children and immunocompromised individuals can develop associated infections, known as BCGitis. Drug susceptibility patterns of BCG clinical strains have rarely been described. We aimed to describe the susceptibility pattern of BCG clinical strains isolated in two different countries. METHODS: We performed culture-based drug susceptibility testing of thirty one BCG strains isolated from patients in Brazil (n=5, 16%) and Argentina (n=26, 84%) using the broth micro-dilution method (phenotypic method). Final antibiotic concentrations for susceptibility testing ranged from 0.5 to 16 mg/L for amikacin, 0.3125 to 10 mg/L for ethambutol, 0.05 to 1.6 mg/L for isoniazid and 0.25 to 8 mg/L for rifampicin, streptomycin and ofloxacin. Additionally, we compared the results with genetic data obtained by whole genome sequencing. RESULTS: By using the phenotypic method we detected one strain resistant to ethambutol, three strains resistant to rifampicin and one resistant to isoniazid. Additionally, two strains that were phenotypically resistant to both isoniazid and rifampicin carried mutations in the katG and rpoB genes simultaneously. CONCLUSION: There is evidence of the emergence of BCG-resistant strains isolated from vaccine-related complications. We recommend drug susceptibility testing of the BCG strain causing the infection in order to prevent treatment failure.

Spatial variation of microtubule depolymerization in large asters.

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.

Characterization of Mycobacterium tuberculosis var. africanum isolated from a patient with pulmonary tuberculosis in Brazil.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.

Computational analysis of filament polymerization dynamics in cytoskeletal networks.

The polymerization-depolymerization dynamics of cytoskeletal proteins play essential roles in the self-organization of cytoskeletal structures, in eukaryotic as well as prokaryotic cells. While advances in fluorescence microscopy and in vitro reconstitution experiments have helped to study the dynamic properties of these complex systems, methods that allow to collect and analyze large quantitative datasets of the underlying polymer dynamics are still missing. Here, we present a novel image analysis workflow to study polymerization dynamics of active filaments in a nonbiased, highly automated manner. Using treadmilling filaments of the bacterial tubulin FtsZ as an example, we demonstrate that our method is able to specifically detect, track and analyze growth and shrinkage of polymers, even in dense networks of filaments. We believe that this automated method can facilitate the analysis of a large variety of dynamic cytoskeletal systems, using standard time-lapse movies obtained from experiments in vitro as well as in the living cell. Moreover, we provide scripts implementing this method as supplementary material.

Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA.

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.