TNF signaling mediates lipopolysaccharide-induced lung epithelial progenitor cell responses in mouse lung organoids.

Bacterial respiratory infections are a major global health concern, often leading to lung injury and triggering lung repair mechanisms. Endogenous epithelial progenitor cells are crucial in this repair, yet the mechanisms remain poorly understood. This study investigates the response of lung epithelial progenitor cells to injury induced by lipopolysaccharide (LPS), a component of gram-negative bacteria, focusing on their regulation during lung repair.

Characterization of NADH fluorescence properties under one-photon excitation with respect to temperature, pH, and binding to lactate dehydrogenase.

Reduced nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolysis and oxidative metabolism and is thus recognized as a key biomarker for probing metabolic state. While the fluorescence characteristics of NADH have been investigated extensively, there are discrepancies in the published data due to diverse experimental conditions, instrumentation and microenvironmental parameters that can affect NADH fluorescence. Using a cuvette-based time-resolved spectrofluorimeter employing one-photon excitation at 375 nm, we characterized the fluorescence intensity, lifetime, spectral response, anisotropy and time-resolved anisotropy of NADH in aqueous solution under varying microenvironmental conditions, namely temperature, pH, and binding to lactate dehydrogenase (LDH).

Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules.

Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size.

The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in vivo results in an increase and decrease, respectively, in EC size and the diameter of microvessels.

Autofluorescence Lifetime Reports Cartilage Damage in Osteoarthritis.

Osteoarthritis (OA) is the most common arthritis and its hallmark is degradation of articular cartilage by proteolytic enzymes leading to loss of joint function. It is challenging to monitor the status of cartilage in vivo and this study explores the use of autofluorescence lifetime (AFL) measurements to provide a label-free optical readout of cartilage degradation that could enable earlier detection and evaluation of potential therapies. We previously reported that treatment of ex vivo porcine cartilage with proteolytic enzymes resulted in decreased AFL.

Epithelioid angiomyolipoma with tumor thrombus.

Epithelioid renal angiomyolipoma is a rare malignant variant of angiomyolipoma (AML); the latter being a well-characterized, benign kidney tumor derived from various connective tissue elements. In this case report, we describe a patient with an epithelioid AML and renal vein thrombus.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells.

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence.

λ = 2.4 - 5 m spectroscopy with the JWST NIRCam instrument.

The James Webb Space Telescope near-infrared camera (JWST NIRCam) has two 2'. 2 × 2'.2 fields of view that can be observed with either imaging or spectroscopic modes.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells.

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis.

Simultaneous imaging of radiation-induced cerebral microbleeds, arteries and veins, using a multiple gradient echo sequence at 7 Tesla.

Background: The purpose of this study was to implement and evaluate the utility of a multi-echo sequence at 7 Tesla (T) for simultaneous time-of-flight (TOF) MR-angiography (MRA) and susceptibility-weighted imaging (SWI) of radiation-induced cerebral microbleeds (CMBs), intracranial arteries, and veins.

Methods: A four-echo gradient-echo sequence was implemented on a 7T scanner. The first echo was used to create TOF-MRA images, while the remaining echoes were combined to visualize CMBs and veins on SWI images.

Remote focal scanning optical projection tomography with an electrically tunable lens.

We describe a remote focal scanning technique for optical projection tomography (OPT) implemented with an electrically tunable lens (ETL) that removes the need to scan the specimen or objective lens. Using a 4× objective lens the average spatial resolution is improved by ∼46% and the light collection efficiency by a factor of ∼6.76, thereby enabling increased acquisition speed and reduced light dose.

Treatment of multiple brain metastases with a divide-and-conquer spatial fractionation radiosurgery approach.

Brain metastases are a common problem, managed with surgery, stereotactic radiosurgery (SRS), whole brain irradiation (WBI), or a combination. SRS targets individual tumors with large dosages of radiation. There is a trend toward using more SRS and less WBI, due to a reduction in cognitive damage, shorter treatment course, and improved tumor control.

The effects of fatigue on robotic surgical skill training in Urology residents.

This study reports on the effect of fatigue on Urology residents using the daVinci surgical skills simulator (dVSS). Seven Urology residents performed a series of selected exercises on the dVSS while pre-call and post-call. Prior to dVSS performance a survey of subjective fatigue was taken and residents were tested with the Epworth Sleepiness Scale (ESS).

Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset).

Intra-articular neurofibroma: an unusual source of anterior knee pain.

The infrapatellar fat pad of the knee is often implicated as a source of anterior knee pain. We present an unusual case of anterior knee pain in an adult patient from a solitary neurofibroma arising in an intra-articular location associated with the infrapatellar fat pad. Clinical, radiographic, magnetic resonance imaging (MRI), and histopathologic features are presented, as well as, a review of the literature.

Detection of cartilage matrix degradation by autofluorescence lifetime.

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix.

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation.

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle.

A novel diagnostic sign of hip fracture mechanism in ground level falls: two case reports and review of the literature.

Introduction: Most elderly hip fractures are the result of a ground level fall. Defining high risk falls and fracture mechanisms are important to develop successful hip fracture prevention programs. This case series presents a previously unreported diagnostic sign and for the first time documents a hip fracture mechanism for a knee impact injury from a ground level fall in two elderly patients.

Face, content, and construct validation of the da Vinci Skills Simulator.

Objective: To report on assessments of face, content, and construct validity for the commercially available da Vinci Skills Simulator (dVSS).

Methods: A total of 38 subjects participated in this prospective study. Participants were classified as novice (0 robotic cases performed), intermediate (1-74 robotic cases), or expert (≥ 75 robotic cases).

A protein biosensor that relies on bending of single DNA molecules.

A "bendy" protein sensor: A DNA-based sensor that uses folded DNA (through DNA kinks) and protein-induced bending to detect DNA-binding proteins is presented. Single-molecule sensing of a transcriptional activator (catabolite activator protein, CAP, which bends its DNA site by 80°) is demonstrated in solution and on surfaces, both in buffers and in cell lysates. The method should allow detection of a wide range of DNA-bending proteins.

Holmium laser enucleation of the prostate technique for benign prostatic hyperplasia.

Holmium laser resection of the prostate (HoLRP) was first described by Gilling et al in 1995. HoLRP has now evolved into holmium laser enucleation of the prostate (HoLEP) with the advent of the intravesical soft-tissue morcellator. The procedure involves anatomical dissection of the prostatic tissue off the surgical capsule in a retrograde fashion using a high-powered holmium laser followed by intravesical morcellation of the prostatic tissue.