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Objective: To assess the correlation between the Visual Analogue Scale (VAS) and the Expanded Prostate Cancer Index Composite (EPIC)-26 in the evaluation of urinary continence (UC) recovery after 3-dimensional laparoscopic radical prostatectomy (3D-LRP)., Methods: 105 men underwent 3D-LRP in Seinäjoki Central Hospital Finland between November 2018 and February 2021. VAS forms and EPIC-26 questionnaires were used to assess UC preoperatively and at 6 weeks, 3-, 6-, 9-, 12-, 15-, 18-, 21-, and 24 months postoperatively. On the VAS form, the patient put a mark on the 10 cm long horizontal line in place, which described his experienced degree of UC (0 cm; fully incontinent-10 cm; fully continent). The scores for the urinary incontinence domain of EPIC-26 (UI-EPIC-26) were calculated and transformed to a scale of 0-100. Spearman´s rank correlation coefficient was used to evaluate the correlation between the VAS and UI-EPIC-26., Results: A total of 915 VAS forms and 909 EPIC-26 questionnaires were evaluable. UC improved significantly during the first year but not after that. The medians for UI-EPIC-26 and VAS were 50.8 (0-100) and 7.2 cm (0-10 cm) at 3 months, 76.8 (14.5-100) and 8.7 cm (1.7-10 cm) at 12 months and 79.6 (8.25-100) and 9.0 cm (2.7-10 cm) at 24 months. The correlation coefficient (95% confidence interval) between VAS and UI-EPIC-26 preoperatively, at 12 months and at 24 months was 0.639 (0.505-0.743), 0.807 (0.716-0.871), and 0.831 (0.735-0.894), respectively (P < 0.001)., Conclusion: The VAS can be utilized as an easy-to-use alternative to the EPIC-26 when evaluating UC recovery after 3D-LRP., Competing Interests: Declaration of Competing Interest Teemu Murtola is paid consultant to Astellas, Amgen, Janssen-Cilag, Novartis, Sanofi, Recordati, Pfizer and Ferring. He reports receiving speaker’s bureau honoraria from Astellas, Amgen, Janssen-Cilag, Merck and Pfizer. He reports congress participation at expense of Sanofi, Orion, and Pfizer. Remaining authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope., (© 2023. The Author(s).)

Objective: To assess suitability of visual analog scale (VAS) in the evaluation of functional outcomes after 3D laparoscopic prostatectomy (3D LRP) METHODS: Two hundred men underwent 3D LRP for localised prostate cancer at Seinäjoki Central Hospital in Finland between December 2013 and September 2018. In October 2019, an EPIC-26 survey along with VAS scales enquiring urinary (VAS-incontinence) and sexual (VAS-sexual) symptoms was mailed to the patients, and the correlations between these 2 methods were evaluated. In the EPIC-26 survey, scores for incontinence-(EPIC-26 UI) and sexual (EPIC-26-sexual) domains were calculated using the University of Michigan scoring system. In the VAS questionnaires, patient put a mark on the 10 cm long horizontal line in place, which described his experience of continence and potency. The Spearman rank correlation coefficient was used to evaluate the correlation between methods., Results: The median scores were as follows: EPIC-26-UI, 79.25 (14.5-100); EPIC-26-sexual, 36.17 (0.0-100); VAS-incontinence, 8.8 cm (1.4-10.0); and VAS-sexual, 3.2 cm (0.0-10). The correlation coefficient between EPIC-26 UI and VAS-incontinence was 0.722 (95% confidence interval [CI], 0.63-0.79; p <.0001) and 0.883 (95% CI, 0.84-0.91; p <.0001) between EPIC-26-sexual and VAS-sexual., Conclusion: Our study shows a strong correlation between VAS and EPIC-26 urinary incontinence and sexual domains. In daily clinical practice VAS-scale may serve as a simple tool to evaluate the key functional outcomes of radical prostatectomy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder. Separate lines of evidence suggest that synapses and astrocytes play a role in the pathological mechanisms underlying ALS. Given that astrocytes make specialised contacts with some synapses, called tripartite synapses, we hypothesise that tripartite synapses could act as the fulcrum of disease in ALS. To test this hypothesis, we have performed an extensive microscopy-based investigation of synapses and tripartite synapses in the spinal cord of ALS model mice and post-mortem human tissue from ALS cases. We reveal widescale synaptic changes at the early symptomatic stages of the SOD1 G93a mouse model. Super-resolution microscopy reveals that large complex postsynaptic structures are lost in ALS mice. Most surprisingly, tripartite synapses are selectively lost, while non-tripartite synapses remain in equal number to healthy controls. Finally, we also observe a similar selective loss of tripartite synapses in human post-mortem ALS spinal cords. From these data we conclude that tripartite synaptopathy is a key hallmark of ALS., (© 2022. The Author(s).)

Immunofluorescence microscopy is routinely used to visualise the spatial distribution of proteins that dictates their cellular function. However, unspecific antibody binding often results in high cytosolic background signals, decreasing the image contrast of a target structure. Recently, convolutional neural networks (CNNs) were successfully employed for image restoration in immunofluorescence microscopy, but current methods cannot correct for those background signals. We report a new method that trains a CNN to reduce unspecific signals in immunofluorescence images; we name this method label2label (L2L). In L2L, a CNN is trained with image pairs of two non-identical labels that target the same cellular structure. We show that after L2L training a network predicts images with significantly increased contrast of a target structure, which is further improved after implementing a multiscale structural similarity loss function. Here, our results suggest that sample differences in the training data decrease hallucination effects that are observed with other methods. We further assess the performance of a cycle generative adversarial network, and show that a CNN can be trained to separate structures in superposed immunofluorescence images of two targets., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations., (© 2021. The Author(s).)

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.

Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We present the first realisation of simultaneous multi-spectral fluorescence imaging using a single-photon avalanche diode (SPAD) array, where the spectral unmixing is facilitated by a plasmonic metasurface mosaic colour filter array (CFA). A 64 × 64 pixel format silicon SPAD array is used to record widefield fluorescence and brightfield data from four biological samples. A plasmonic metasurface composed of an arrangement of circular and elliptical nanoholes etched into an aluminium thin film deposited on a glass substrate provides the high transmission efficiency CFA, enabling a bespoke spectral unmixing algorithm to reconstruct high fidelity, full colour images from as few as ∼3 photons per pixel. This approach points the way toward real-time, single-photon sensitive multi-spectral fluorescence imaging. Furthermore, this is possible without additional bulky components such as a filter wheel, prism or diffraction grating, nor the need for multiple sample exposures or multiple detectors., (© 2021 The Authors. Journal of Biophotonics published by Wiley-VCH GmbH.)

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies., Competing Interests: The authors have declared that no competing interests exist.

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens., Competing Interests: We have no competing interests.

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Naïve CD4+ T cells may differentiate into a number of subsets including T helper 1 (Th1) Th2, Th3, Th17 and T regulatory (Treg) cells depending on the type of antigen they encounter. These CD4+ families have been defined based on the array of cytokines they produce and the effects they have on adaptive immune responses. CD4+ subsets are cross regulatory and at times cooperative. The study of these adaptive immune modulators has revealed the important role that cytokines play in mounting effective as well as detrimental immune responses to pathogens. Examining the cytokine responses of lymphocytes in culture can provide important understanding of how immune responses to pathogens are orchestrated. For this purpose the in vitro cytokine production of peripheral blood mononuclear cells (PBMC) from healthy dogs was examined in response to stimulation with antigens from a common canine virus (canine distemper virus, CDV), a commensal skin yeast of dogs (Malassezia pachydermatis) and a common canine helminth (Toxocara canis (T. canis)). Cell culture supernatants were removed from antigen stimulated and unstimulated control PBMC after 4, 24, 48 and 72 h and the concentration of Th1 type cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 type cytokines (IL-4, IL-5, IL-10) was determined using sandwich ELISA assays. CDV induced low levels of cytokine production initially with a predominance of IL-10 at 24h and a balanced response at 48 h of incubation. Malassezia antigen stimulated an early type 2 cytokine response with dramatic production of IL-4 at 24h of incubation compared to the other stimulants examined. By 48 h of incubation, however, the cytokine mix in response to Malassezia had also moved toward a Th1 type response. T. canis induced early production of Th2 type cytokines with IL-5 predominating; however, with longer incubation (48-72 h) there was a switch to a balanced Th1/Th2 response. In conclusion, the cytokines produced in vitro by canine PBMC in response to prototypical Th1 and Th2 type pathogens were not clearly polarized and shifted over time. While the in vitro study of PBMC cytokine responses cannot be directly extrapolated to in vivo responses to the same antigens, the results do highlight the dynamic and fluctuating nature of cytokine production., (Copyright 2009 Elsevier B.V. All rights reserved.)

Objective: To discuss the treatment of a patient with type 1 diabetes presenting with chronic neck and shoulder pain by using chiropractic manipulation and an active rehabilitation program with emphasis on correcting postural imbalances., Clinical Features: A 46-year-old insulin dependant (type1) diabetic female presented with neck and right shoulder pain of 6 to 8 months duration. Her history included similar left-sided complaints 2 years prior at which time she underwent 3 months of rehabilitation at a local medical center, which improved her condition. Over time her pain resolved but the residuals of restricted left shoulder range of motion remained. The patient had postural changes consisting of forward head posture, rounded shoulders and internally rotated arms., Intervention and Outcome: Treatment included spinal manipulation, ultrasound and active rehabilitation consisting of at home exercises initially and followed with in office low-tech rehabilitation. Rehabilitation was primarily aimed at improving postural abnormalities, muscle imbalances and abnormal movement patterns. The patient improved with this course of treatment., Conclusion: Chiropractic care including active rehabilitation may be helpful in treating diabetic patients suffering from chronic neck and shoulder problems.

A continuous infusion of propofol following an induction dose of 2 mg/kg was compared with thiopental/isoflurane for the induction and maintenance of anesthesia in 20 mentally retarded outpatients undergoing routine dental procedures. The infusion rate of propofol and the concentration of isoflurane were adjusted to maintain the heart rate and blood pressure within +/- 25% of the baseline values. Postoperative wakefulness was assessed using a 100-mm visual analogue scale at the time of extubation and at 5, 10, 15, 30, 60, 90, and 120 min after extubation. Both agents provided adequate anesthesia for the treatment, and no major adverse reactions occurred. Recovery was more complete during the first hour after extubation in the propofol group, and these patients were discharged earlier.