Miracle-dz/UniFAR-FTUs huggingface dataset:
Quantification and its Applications in Fluorescent Microscopy Imaging

Fluorescent microscope imaging technologies have developed at a rapid pace in recent years. High‐throughput 2D fluorescent imaging platforms are now in wide use and are being applied on a proteome wide scale. Multiple fluorophore 3D imaging of live cells is being used to give detailed localization and subcellular structure information. Further, 2D and 3D video microscopy are giving important insights into the dynamics of protein localization and transport. In parallel with these developments, significant research has gone into developing new methodologies for quantifying and extracting meaning from the imaging data. Here we outline and give entry points to the literature on approaches to quantification such as segmentation, tracking, automated classification and data visualization. Particular attention is paid to the distinction between and application of concrete quantification measures such as number of objects in a cell, and abstract measures such as texture.
Peroxisome Dynamics in Cultured Mammalian Cells

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein–a mutated bacterial dehalogenase which is fused to the protein of interest–and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell‐permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag‐containing construct, at different wavelengths. Here, we show that peroxisomes display an age‐related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre‐existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half‐life of approximately 2 days and are mainly degraded by an autophagy‐related mechanism. The implications of these findings are discussed.
Standardization and documentation of varicocele evaluation

Purpose of review Our present understanding of the clinical impact of varicocele on male fertility and the efficacy of varicocele treatment is limited by the absence of an objective, reproducible, and standardized diagnostic evaluation. Herein we review the clinical evaluation of varicocele. The prognostic relevance of varicocele grade and venous diameter measured with ultrasound is explored. Recent findings A directed history, physical examination of the warmed scrotum, and results of one or multiple semen analyses must be documented in all men with varicoceles. Color Doppler ultrasonography is indicated in cases when the physical examination is indeterminate. Unfortunately, physical examination is limited by intraobserver and interobserver bias, and standardized criteria for the ultrasonographic diagnosis of varicocele do not exist. Despite these limitations, both varicocele grade and venous diameter measured on ultrasound are prognostically useful parameters. In select cases, measurement of serum testosterone and assessment of sperm DNA integrity may also be clinically helpful. Summary The absence of standardized, reproducible clinical, and radiographic evaluations for varicocele has contributed substantially to our present difficulties in selecting patients who are likely to benefit from varicocele treatment and in counseling affected men. Consensus regarding the optimal evaluation of varicoceles and widespread acceptance of a standardized evaluation is necessary.
Implications of changing the Six‐item Cognitive Impairment Test cutoff

The Six-itemCognitive Impairment Test (6CIT; Brooke and Bullock, 1999), a brief (2–3min) cognitive screening instrument, was administered to consecutive new outpatients attending a dedicated cognitive disorders clinic over a 12-month period (Abdel-Aziz and Larner, 2015). In this study, 6CIT scores were classified for test interpretation as per Jefferies and Gale (2013): normal cognition (0–4), questionable impairment (5–9), and suggesting impairment consistent with dementia and requiring further evaluation (10–28). At the specified cutoff>4, sensitivity and specificity for diagnosis of dementia versus no dementia were 0.88 and 0.78, respectively; at the cutoff >9, sensitivity and specificity for diagnosis of mild cognitive impairment (MCI) versus no cognitive impairment were 0.66 and 0.70, respectively (Abdel-Aziz and Larner, 2015). Other sources report different 6CIT score ranges, namely 0–7 normal and ≥8 significant (www.patient. co.uk/doctor/six-item-cognitive-impairment-test-6cit), with stated overall sensitivity and specificity of 90% and 100% and for “mild dementia” (not defined) of 78% and 100%. Experience in this clinic indicates that 6CIT 7/8 threshold is now being used in some primary care settings as criterion for onward referral of patients for further assessment. Changes in test cutoff may have significant implications for measures of test discrimination such as sensitivity and specificity (Larner, 2015). Accordingly, the original study dataset was reinterrogated to quantify test metrics at 6CIT 7/8 cutoff. It was hypothesized that the now higher cutoff for dementia (7 vs. 4, =lower threshold, 6CIT negatively scored) would be more sensitive but less specific than the previously examined cutoff; whereas the now lower cutoff for MCI (7 vs. 9, =higher threshold) would be less sensitive but more specific. Of 245 patients assessed (F :M=121:124, 49.4% women; age range 16–94years; median 59years), 48 were diagnosed with dementia (prevalence 19.6%) and a further 67 (27.3%) had MCI. Screening utility of 6CIT for all the various diagnostic comparisons (dementia vs. no dementia, n=245; dementia vs. MCI, n=115; MCI vs. normal cognition/subjective memory impairment, n=197) was calculated at the cutoff ≥8 (Tables 1 and 2) and compared with the outcomes of previously used cutoffs for dementia (>4) and MCI (>9). The findings confirmed the anticipated greater sensitivity of the higher cutoff for dementia (0.90 vs. 0.88) with lower specificity (0.68 vs. 0.78) and lower sensitivity of the lower cutoff for MCI (0.55 vs. 0.66) with Table 1 Demographic parameters for 6CIT for diagnosis of dementia
Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long‐distance transport can be studied by acquiring fast time‐lapse images of green fluorescent protein (GFP)‐tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high‐resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.
Ezrin Promotes Actin Assembly at the Phagosome Membrane and Regulates Phago‐Lysosomal Fusion

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F‐actin at their membrane, and that the ezrin‐radixin‐moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N‐WASP (Neural Wiskott‐Aldrich Syndrome Protein) by its FERM domain. Using a cell‐free system, we found that ezrin stimulates F‐actin assembly on purified phagosomes by recruiting the N‐WASP–Arp2/3 machinery. Accordingly, we showed that the down‐regulation of ezrin activity in macrophages by a dominant‐negative approach caused reduced F‐actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live‐cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.
A Study of Saliva and Its Action on Tooth Enamel in Reference to Its Hardening and Softening

Enamel softening has been considered to be necessarily associated with roughening of the surface, loss of luster and dissolution of the cemental substance that binds the enamel rods together; and whenever enamel decalcification is associated with decay of dentin this conception of enamel softening is only too accurate. But this conception seems to be only a part of the story, so to speak—the final part rather than the complete process of enamel degeneration. For instance, 1 to 1,000 lactic acid and water at mouth temperature will cut tooth enamel in thirty minutes with a rough, white surface. A tooth placed in 1 to 500 lactic acid and some salivas will be unharmed. A tooth placed in this solution made with other salivas, after three or four days, may show the enamel perfectly smooth and to all appearances normal, and yet it can readily be pared to a slight
Cysteine Ubiquitination of PTS1 Receptor Pex5p Regulates Pex5p Recycling

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome‐targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine11 in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p‐C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild‐type cells. Pex5p‐C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N‐terminal 158‐amino‐acid region of Pex5p‐C11A, termed 158‐CA, is sufficient for such dominant‐negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p‐C11A or 158‐CA likewise inhibits the wild‐type Pex5p import into peroxisomes, strongly suggesting that Pex5p‐C11A exerts the dominant‐negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine11 of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p‐C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.
The Gnosall project: Setting new benchmarks for dementia care

A pioneering project in Staffordshire is expected to play a pivotal role in shaping the treatment of dementia in the UK. The GP who developed the scheme has already won national recognition for his work. Dr Ian Greaves received the 2010 GP Enterprise Award from the Royal College of Practitioners. He introduced a service at the Gnosall Health Centre that helped to reduce the diagnosis period for dementia from the national average of 3 years to just 4 weeks. Since its launch, the project has saved the NHS about £400,000. The National Dementia Strategy recognises the importance of early diagnosis and improved patient care. This paper describes how specialist services, families, and primary care providers have worked together to make the project a success — particularly through the screening of primary care vascular databases for patients with cognitive decline. There is considerable evidence associating vascular disease and dementia.
The Ins and Outs of HIV‐1 Tat

HIV‐1 encodes for the small basic protein Tat (86–101 residues) that drastically enhances the efficiency of viral transcription. The mechanism enabling Tat nuclear import is not yet clear, but studies using reporter proteins fused to the Tat basic domain indicate that Tat could reach the nucleus by passive diffusion. Tat also uses an unusual transcellular transport pathway. The first step of this pathway involves high‐affinity binding of Tat to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), a phospholipid that is concentrated in the inner leaflet of the plasma membrane and enables Tat recruitment at this level. Tat then crosses the plasma membrane to reach the outside medium. Although unconventional, Tat secretion by infected cells is highly active, and export is the major destination for HIV‐1 Tat. Secreted Tat can bind to a variety of cell types using several different receptors. Most of them will allow Tat endocytosis. Upon internalization, low endosomal pH triggers a conformational change in Tat that results in membrane insertion. Later steps of Tat translocation to the target‐cell cytosol are assisted by Hsp90, a general cytosolic chaperone. Cytosolic Tat can trigger various cell responses. Indeed, accumulating evidence suggests that extracellular Tat acts as a viral toxin that affects the biological activity of different cell types and has a key role in acquired immune‐deficiency syndrome development. This review focuses on some of the recently identified molecular details underlying the unusual transcellular transport pathway used by Tat, such as the role of the single Trp in Tat for its membrane insertion and translocation.
A Centronuclear Myopathy – Dynamin 2 Mutation Impairs Autophagy in Mice

Dynamin 2 (Dnm2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous (HTZ) mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot–Marie–Tooth neuropathy. We generated a knock‐in Dnm2R465W mouse model expressing the most frequent human mutation and recently reported that HTZ mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous (HMZ) mice. We show that HMZ mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in HMZ embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved HMZ cells have a higher number of immature autophagy‐related structures probably due to a defect of acidification. Our results highlight the role of Dnm2 in the cross talk between endosomal and autophagic pathways and evidence a new role of Dnm2‐dependent membrane trafficking in autophagy which may be relevant in DNM2‐related human diseases.
Direct Endoscopic Necrosectomy Versus Step-Up Approach for Walled-Off Pancreatic Necrosis: Comparison of Clinical Outcome and Health Care Utilization

Objectives Infected walled-off pancreatic necrosis (WOPN) is a complication of acute pancreatitis requiring intervention. Surgery is associated with considerable morbidity. Percutaneous catheter drainage (PCD), initial therapy in the step-up approach, minimizes complications. Direct endoscopic necrosectomy (DEN) has demonstrated safety and efficacy. We compared outcome and health care utilization of DEN versus step-up approach. Methods This was a matched cohort study using a prospective registry. Twelve consecutive DEN patients were matched with 12 step-up approach patients. Outcomes were clinical resolution after primary therapeutic modality, new organ failure, mortality, endocrine or exocrine insufficiency, length of stay, and health care utilization. Results Clinical resolution in 11 of 12 patients after DEN versus 3 of 12 step-up approach patients after PCD (P < 0.01). Nine step-up approach patients required surgery; 7 of these experienced complications. Direct endoscopic necrosectomy resulted in less new antibiotic use, pulmonary failure, endocrine insufficiency, and shorter length of stay (P < 0.05). Health care utilization was lower after DEN by 5.2:1 (P < 0.01). Conclusions Direct endoscopic necrosectomy may be superior to step-up approach for WOPN with suspected or established infection. Primary PCD generally delayed definitive therapy. Given the higher efficacy, shorter length of stay, and lower health care utilization, DEN could be the first-line therapy for WOPN, with primary PCD for inaccessible or immature collections.
New composites of betulin esters with arabinogalactan as highly potent anti-cancer agents

Abstract Betulin and its esters are the natural compounds with high in vitro cytotoxicity toward many cancer cells. However, the poor water solubility of these compounds has limited their applications. We prepared new composites of betulin esters using two methods, namely ball-milling of the mixtures of betulin esters with arabinogalactan and preparation of thin films of these mixtures by evaporating the aqueous solutions. These composites revealed higher water solubility as compared with the initial substances without losing the structural integrity and functionality. As a result, the new composites have shown much higher inhibitory effects against different cancer cell lines such as Ehrlich ascites carcinoma cells and lung carcinoma cells (A549) in comparison with the initial substances. The cell viability studies based on Annexin V and Propidium iodide probes have confirmed the high proapoptotic effect of betulin ester derivatives against cancer cells. Graphical abstract
Circulating Tumor Cells Found in Patients With Localized and Advanced Pancreatic Cancer

Objectives Isolation of circulating tumor cells (CTCs) holds the promise of diagnosing and molecular profiling cancers from a blood sample. Here, we test a simple new low-cost filtration device for CTC isolation in patients with pancreatic ductal adenocarcinoma (PDAC). Methods Peripheral blood samples drawn from healthy donors and PDAC patients were filtered using ScreenCell devices, designed to capture CTCs for cytologic and molecular analysis. Giemsa-stained specimens were evaluated by a pancreatic cytopathologist blinded to the histological diagnosis. Circulating tumor cell DNA was subjected to KRAS mutational analysis. Results Spiking experiments demonstrated a CTC capture efficiency as low as 2 cells/mL of blood. Circulating tumor cells were identified by either malignant cytology or presence of KRAS mutation in 73% of 11 patients (P = 0.001). Circulating tumor cells were identified in 3 of 4 patients with early (⩽American Joint Committee on Cancer stage IIB) and in 5 of 7 patients with advanced (≥ American Joint Committee on Cancer stage III) PDAC. No CTCs were detected in blood from 9 health donors. Conclusions Circulating tumor cells can be found in most patients with PDAC of any stage, whether localized, locally advanced, or metastatic. The ability to capture, cytologically identify, and genetically analyze CTCs suggests a possible tool for the diagnosis and characterization of genetic alterations of PDAC.
Dalvelutinoside, a new isoflavone glycoside from the methanol extract of Dalbergia velutina roots

Abstract A new isoflavone glycoside, dalvelutinoside (1), together with one known isoflavone (2) and five known isoflavone glycosides (3–7) were isolated from the methanol extract of the roots of Dalbergia velutina. Their structures were determined by spectroscopic analysis. All isolated compounds were evaluated for their cytotoxicity against KB and HeLa cell lines.
Biotransformation of ursolic acid by Alternaria longipes AS3.2875

Abstract Microbial transformation of ursolic acid (1) was carried out by Alternaria longipes AS 3.2875. Six transformed products (2–7) from 1 were isolated and their structures were identified as 3-carbonyl ursolic acid 28-O-β-D-glucopyranosyl ester (2), ursolic acid 3-O-β-D-glucopyranoside (3), ursolic acid 28-O-β-D-glucopyranosyl ester (4), 2α, 3β-dihydroxy ursolic acid 28-O-β-D-glucopyranosyl ester (5), 3β, 21β dihydroxy ursolic acid 28-O-β-D-glucopyranosyl ester (6), and 3-O-(β-D-glucopyranosyl)- ursolic acid 28-O-(β-D-glucopyranosyl) ester (7) based on the analysis of 1D NMR, 2DNMR and MS data. The product 2 was a new compound among them and showed stronger antibacterial activity against S. aureu, MRSA and MRCA than substrate. In this study, we modified structure of ursolic acid through biotransformation to enhance its activities and preliminarily discussed the transformation way of the products.
Somatosensory system in two types of motor reorganization in congenital hemiparesis: Topography and function

This study investigates the (re‐)organization of somatosensory functions following early brain lesions. Using functional magnetic resonance imaging (fMRI), passive hand movement was studied. Transcranial magnetic stimulation (TMS) and magnetoencephalography (MEG) were used as complementary methods. fMRI data was analyzed on the first level with regard to topographical variability; second‐level group effects as well as the overall integrity of the somatosensory circuitry were also assessed. Subjects with unilateral brain lesions occurring in the third trimester of pregnancy or perinatally with different types of motor reorganization were included: patients with regular, contralateral motor organization following middle cerebral artery strokes (CONTRAMCA, n = 6) and patients with reorganized, ipsilateral motor functions due to periventricular lesions (IPSIPL, n = 8). Motor impairment was similar, but sensory impairment was more pronounced in the CONTRAMCA group. Using fMRI and MEG, both groups showed a normal pattern with a contralateral somatosensory representation, despite the transhemispherically reorganized primary motor cortex in the IPSIPL group, as verified by TMS. Activation topography for the paretic hands was more variable than for the nonparetic hand in both groups. The cortico‐cerebellar circuitry was well‐preserved in almost all subjects. We conclude that in both models of motor reorganization, no interhemispheric reorganization of somatosensory functions occurred. Also, no relevant intrahemispheric reorganization was observed apart from a higher topographical variability of fMRI activations. This preserved pattern of somatosensory organization argues in favor of a differential lesion effect on motor and somatosensory functions and demonstrates a limited compensatory potential for the latter. Hum Brain Mapp, 2009. © 2008 Wiley‐Liss, Inc.