Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease.

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.

Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism.

We report the use of clickable monoacylglycerol (MAG) analogs as probes for the labeling of glycerolipids during lipid metabolism. Incorporation of azide tags onto the glycerol region was pursued to develop probes that would label glycerolipids, in which the click tag would not be removed through processes including acyl chain and headgroup remodeling. Analysis of clickable MAG probes containing acyl chains of different length resulted in widely variable cell imaging and cytotoxicity profiles. Based on these results, we focused on a probe bearing a short acyl chain (C4 -MAG-N3 ) that was found to infiltrate natural lipid biosynthetic pathways to produce click-tagged versions of both neutral and phospholipid products. Alternatively, strategic blocking of the glycerol sn-3 position in probe C4 -MEG-N3 served to deactivate phospholipid tagging and focus labeling on neutral lipids. This work shows that lipid metabolic labeling profiles can be tuned based on probe structures and provides valuable tools for evaluating alterations to lipid metabolism in cells.

Contact-Free, Passive, Electromagnetic Resonant Sensors for Enclosed Biomedical Applications: A Perspective on Opportunities and Challenges.

Inexpensive and accurate tools for monitoring conditions in enclosed environments (through garments, bandages, tissue, etc.) have been a long-standing goal of medicine. Passive resonant sensors are a promising solution for such wearable health sensors as well as off-body diagnostics. They are simple circuits with inherent inductance and capacitance (LC tank) that have a measurable resonant frequency. Changes in local parameters, e.g., permittivity or geometry, effect inductance and capacitance which cause a resonant frequency shift response. This signal transduction has been applied to several biomedical applications such as intracranial pressure, hemodynamics, epidermal hydration, etc. Despite these many promising applications presented in the literature, resonant sensors still do not see widespread adoption in biomedical applications, especially as wearable or embedded sensing devices. This perspective highlights some of the current challenges facing LC resonant sensors in biomedical applications, such as positional sensitivity, and potential strategies that have been developed to overcome them. An outlook on adoption in medicine and health monitoring is presented, and a perspective is given on next steps for research in this field.

Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes.

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-l-SerN3) or carbonyl (C-l-SerN3) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.

Rapid Characterization of Solid Tumors Using Resonant Sensors.

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors.

Toward Mail-in-Sensors for SARS-CoV-2 Detection: Interfacing Gel Switch Resonators with Cell-Free Toehold Switches.

The COVID-19 pandemic has emphasized the importance of widespread testing to control the spread of infectious diseases. The rapid development, scale-up, and deployment of viral and antibody detection methods since the beginning of the pandemic have greatly increased testing capacity. Desirable attributes of detection methods are low product costs, self-administered protocols, and the ability to be mailed in sealed envelopes for the safe analysis and subsequent logging to public health databases. Herein, such a platform is demonstrated with a screen-printed, inductor-capacitor (LC) resonator as a transducer and a toehold switch coupled with cell-free expression as the biological selective recognition element. In the presence of the N-gene from SARS-CoV-2, the toehold switch relaxes, protease enzyme is expressed, and it degrades a gelatin switch that ultimately shifts the resonant frequency of the planar resonant sensor. The gelatin switch resonator (GSR) can be analyzed through a sealed envelope allowing for assessment without the need for careful sample handling with personal protective equipment or the need for workup with other reagents. The toehold switch used in this sensor demonstrated selectivity to SARS-CoV-2 virus over three seasonal coronaviruses and SARS-CoV-1, with a limit of detection of 100 copies/µL. The functionality of the platform and assessment in a sealed envelope with an automated scanner is shown with overnight shipment, and further improvements are discussed to increase signal stability and further simplify user protocols toward a mail-in platform.

"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Monitoring Wound Health through Bandages with Passive LC Resonant Sensors.

This paper details a passive, inductor-capacitor (LC) resonant sensor embedded in a commercial dressing for low-cost, contact-free monitoring of a wound; this would enable tracking of the healing process while keeping the site closed and sterile. Spiral LC resonators were fabricated from flexible, copper-coated polyimide and interrogated using external reader antennas connected to a two-port vector network analyzer; the forward transmission scattering parameter (S21) magnitude was collected, and the resonant frequency (MHz) and the peak-to-peak amplitude of the resonant feature were identified. These increase during the healing process as the permittivity and conductivity of the tissue change. The sensor was first tested on gelatin-based tissue-mimicking phantoms that simulate layers of muscle, blood, fat, and skin at varying phases of wound healing. Finite element modeling was also used to verify the empirical results based on the expected variations in dielectric properties of the tissue. The performance of the resonant sensors for in vivo applications was investigated by conducting animal studies using canine patients that presented with a natural wound as well as a controlled cohort of rat models with surgically administered wounds. Finally, transfer functions are presented that relate the resonant frequency to wound size using an exponential model (R2 = 0.58-0.96). The next steps in sensor design and fabrication as well as the reading platform to achieve the goal of a universal calibration curve are then discussed.

A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC-γ Isozymes Operating at Membranes.

The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.

Sweat monitoring beneath garments using passive, wireless resonant sensors interfaced with laser-ablated microfluidics.

Sweat loss can help determine hydration status of individuals working in harsh conditions, which is especially relevant to those who wear thick personal protective equipment (PPE) such as firefighters. A wireless, passive, conformable sweat sensor sticker is described here that can be worn under and interrogated through thick clothing to simultaneously measure sweat loss volume and conductivity. The sticker consists of a laser-ablated, microfluidic channel and a resonant sensor transducer. The resonant sensor is wirelessly read with a handheld vector network analyzer coupled to two, co-planar, interrogation antennas that measure the transmission loss. A sweat proxy is used to fill the channels and it is determined that the sensor can orthogonally determine the sweat conductivity and volume filled in the channel via peak transmission loss magnitude and frequency respectively. A four-person study is then used to determine level of sensor variance caused by local tissue dielectric heterogeneity and sensor-reader orientation.

Clinical Implications of a One-hand Versus Two-hand Technique in the Silfverskiöld Test for Gastrocnemius Equinus.

Introduction Isolated gastrocnemius equinus contracture has been associated with several foot and ankle pathologies within the literature. The Silfverskiöld test is commonly used to identify isolated gastrocnemius contracture, however, the proper technique for performing the test has been scrutinized. The purpose of this study was to determine if there is a clinical significance in the ankle dorsiflexion that is obtained when the examination is performed incorrectly with a single hand versus the correct two-hand technique. Methods Thirty consecutive new patients with conditions associated with gastrocnemius equinus were included in the study. The Silfverskiöld test was performed with a two-hand technique and a single-hand technique. The amount of dorsiflexion obtained with the knee in full extension was measured and recorded using an extendable goniometer for each technique, with the arms aligned with the fifth metatarsal and fibular head. Results The average amount of dorsiflexion that was obtained with the two-hand technique with the knee in full extension was 76.3°±4.2°. When the one-hand technique was utilized the average amount of dorsiflexion obtained with the knee in full extension was 88.4°±4.2°. This was found to be statistically significant (p<0.01). Conclusion This study demonstrates that if the Silfverskiöld test is not performed correctly, the diagnosis of an isolated gastrocnemius contracture could be underappreciated. Accordingly, it may be important to perform the test with two hands in order to neutralize the hindfoot, midfoot, and forefoot, so that the dorsiflexion motion is through the tibiotalar joint alone.

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

Tandem Specialty Management of Complex Lower Extremity Wounds: A Report of Three Cases.

Complex lower extremity wounds present a unique problem to foot and ankle clinicians, with many obstacles to achieving a successful outcome. The decreased vasculature of the lower extremities creates environments where wounds lack the resources to properly heal on their own. Conditions such as diabetes mellitus and smoking can exacerbate these issues by further decreasing vascular flow providing resources to the wound. For physicians trained in orthopedic foot and ankle surgery, they often do not receive training in advanced wound care, whereas podiatric surgeons can obtain fellowship training in wound care management. This dynamic presents a unique opportunity for tandem management of complex lower extremity wounds, which can decrease patient morbidity and the costs associated with care. We present three cases of complex wounds managed in a tandem fashion that achieved optimal outcomes after both orthopedic surgery and podiatric surgery were involved. These cases illustrate the potential benefits associated with tandem wound management in foot and ankle surgery.

Calcium-Responsive Liposomes via a Synthetic Lipid Switch.

Liposomal drug delivery would benefit from enhanced control over content release. Here, we report a novel avenue for triggering release driven by chemical composition using liposomes sensitized to calcium-a target chosen due to its key roles in biology and disease. To demonstrate this principle, we synthesized calcium-responsive lipid switch 1, designed to undergo conformational changes upon calcium binding. The conformational change perturbs membrane integrity, thereby promoting cargo release. This was shown through fluorescence-based release assays via dose-dependent response depending on the percentage of 1 in liposomes, with minimal background leakage in controls. DLS experiments indicated dramatic changes in particle size upon treatment of liposomes containing 1 with calcium. In a comparison of ten naturally occurring metal cations, calcium provided the greatest release. Finally, STEM images showed significant changes in liposome morphology upon treatment of liposomes containing 1 with calcium. These results showcase lipid switches driven by molecular recognition principles as an exciting avenue for controlling membrane properties.

Artificial Membrane Fusion Triggered by Strain-Promoted Alkyne-Azide Cycloaddition.

Artificial systems for controlled membrane fusion applicable for drug delivery would ideally use triggers that are orthogonal to biology. To apply the strain-promoted alkyne-azide cycloaddition (SPAAC) to drive membrane fusion, oxo-dibenzocyclooctyne (ODIBO)-lipid 1 was designed, synthesized, and studied alongside azadibenzocyclooctyne (ADIBO)-lipids 2-4 to assess fusion with liposomes containing azido-lipid 5. Lipids 1-2 were first shown to be effective for liposome derivatization. Next, fusion was evaluated using liposomes containing 1 and varying ratios of PC and PE via a FRET dilution fusion assay, and a 1:1 PC-to-PE ratio yielded the greatest signal change attributed to fusion. Finally, lipids 1-4 were compared, and 1 yielded the greatest triggering of fusion, while 2-4 yielded varying efficacies depending on the structural features of each lipid. Fusion was further validated through STEM studies showing larger multilamellar assemblies after liposome mixing, and FRET assay results supporting the mixing of liposome aqueous contents. This work provides a platform for triggered fusion toward drug delivery applications and an understanding of the effects of lipid structure and membrane composition on fusion.

Particle formation of budesonide from alcohol-modified subcritical water solutions.

Recently, subcritical water (SBCW: water that has been heated to a temperature between 100°C and 200°C at pressures of up to 70bar) has been used to dissolve several hydrophobic pharmaceutical compounds (Carr et al., 2010a). Furthermore, a number of active pharmaceutical ingredients (APIs) have been rapidly precipitated from SBCW solutions (Carr et al., 2010b,c). It is possible to alter the precipitate morphology by altering the processing variables; including the SBCW-API solution injection temperature and adding impurities (such as pharmaceutical excipients, e.g. lactose) to the precipitation chamber. The work presented in this article demonstrates that the morphology of pharmaceutical particles can be tuned by adding organic solvents (ethanol and methanol) to the SBCW-API solutions. Particle morphology has also been tuned by adding different pharmaceutical excipients (polyethylene glycol 400 and lactose) to the precipitation chamber. Different morphologies of pharmaceutical particles were produced, ranging from nanospheres of 60nm diameter to 5µm plate particles. Budesonide was used as the model API in this study. Two experimental products were spray dried to form dry powder products. The aerodynamic particle size of the powder was established by running the powder through an Andersen Cascade Impactor. It has been shown that the drug particles produced from the SBCW micronization process, when coupled with a spray drying process, are suitable for delivery to the lungs.