The antisickling agent, 5-hydroxymethyl-2-furfural: Other potential pharmacological applications.

For the last two decades, the aromatic aldehyde 5-hydroxymethyl-furfural (5-HMF) has been the subject of several investigations for its pharmacologic potential. In 2004, the Safo group reported that 5-HMF has potent antisickling activity by targeting and ameliorating the primary pathophysiology of hypoxia-induced sickling of erythrocytes (red blood cells [RBC]). Following the encouraging outcome of the preclinical and phase I/II clinical studies of 5-HMF for the treatment of sickle cell disease (SCD), there have been multiple studies suggesting 5-HMF has several other biological or pharmacologic activities, including anti-allergic, antioxidant, anti-hypoxic, anti-ischemic, cognitive improvement, anti-tyrosinase, anti-proliferation, cytoprotective, and anti-inflammatory activities. The wide range of its effects makes 5-HMF a potential candidate for treating a variety of diseases including cognitive disorders, gout, allergic disorders, anemia, hypoxia, cancers, ischemia, hemorrhagic shock, liver fibrosis, and oxidative injury. Several of these therapeutic claims are currently under investigation and, while promising, vary in terms of the strength of their evidence. This review presents the research regarding the therapeutic potential of 5-HMF in addition to its sources, physicochemical properties, safety, absorption, distribution, metabolism, and excretion (ADME) profiles.

Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation.

Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay - quantifying the appearance of Hb-AEH adduct - as a "universal" method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.

Metabolic Reprogramming in Sickle Cell Diseases: Pathophysiology and Drug Discovery Opportunities.

Sickle cell disease (SCD) is a genetic disorder that affects millions of individuals worldwide. Chronic anemia, hemolysis, and vasculopathy are associated with SCD, and their role has been well characterized. These symptoms stem from hemoglobin (Hb) polymerization, which is the primary event in the molecular pathogenesis of SCD and contributes to erythrocyte or red blood cell (RBC) sickling, stiffness, and vaso-occlusion. The disease is caused by a mutation at the sixth position of the ß-globin gene, coding for sickle Hb (HbS) instead of normal adult Hb (HbA), which under hypoxic conditions polymerizes into rigid fibers to distort the shapes of the RBCs. Only a few therapies are available, with the universal effectiveness of recently approved therapies still being monitored. In this review, we first focus on how sickle RBCs have altered metabolism and then highlight how this understanding reveals potential targets involved in the pathogenesis of the disease, which can be leveraged to create novel therapeutics for SCD.

Mechanistic Analysis of the VirA Sensor Kinase in Agrobacterium tumefaciens Using Structural Models.

Agrobacterium tumefaciens pathogenesis of plants is initiated with signal reception and culminates with transforming the genomic DNA of its host. The histidine sensor kinase VirA receives and reacts to discrete signaling molecules for the full induction of the genes necessary for this process. Though many of the components of this process have been identified, the precise mechanism of how VirA coordinates the response to host signals, namely phenols and sugars, is unknown. Recent advances of molecular modeling have allowed us to test structure/function predictions and contextualize previous experiments with VirA. In particular, the deep mind software AlphaFold has generated a structural model for the entire protein, allowing us to construct a model that addresses the mechanism of VirA signal reception. Here, we deepen our analysis of the region of VirA that is critical for phenol reception, model and probe potential phenol-binding sites of VirA, and refine its mechanism to strengthen our understanding of A. tumefaciens signal perception.

Vaccines for COVID-19.

Since the emergence of COVID-19, caused by the SARS-CoV-2 virus at the end of 2019, there has been an explosion of vaccine development. By 24 September 2020, a staggering number of vaccines (more than 200) had started preclinical development, of which 43 had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. Vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socializing. Importantly, to effectively control the COVID-19 pandemic, production needs to be scaled-up from a small number of preclinical doses to enough filled vials to immunize the world's population, which requires close engagement with manufacturers and regulators. It will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. This review explores the immune responses required to protect against SARS-CoV-2 and the potential for vaccine-induced immunopathology. We describe the profile of the different platforms and the advantages and disadvantages of each approach. The review also addresses the critical steps between promising preclinical leads and manufacturing at scale. The issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. Nine months after the outbreak began we are at a point where preclinical and early clinical data are being generated for the vaccines; an overview of this important area will help our understanding of the next phases.

Mapping Reaction-Diffusion Networks at the Plant Wound Site With Pathogens.

The rich collection of microbes colonizing the plant root making up the rhizosphere function as a multigenomic organ for nutrient distribution. The extent to which its dynamic mutualistic cellular order depends on morphogenic signaling, while likely, remains unknown. We have shown that reaction-diffusion chemical networks constructed with model plant and bacterial metabolites can mimic processes ranging from oxidative burst kinetics to traveling waves and extracellular stationary state reaction-diffusion networks for spatiotemporal ordering of the rhizosphere. Plant parasites and pathogens can be limited by host attachment require dynamic informational networks and continue to provide insight into what controls the rhizosphere. Here we take advantage of Agrobacterium tumefaciens, a plant pathogen with a gated receptor that requires simultaneous perception of two plant metabolites. Genetic manipulations have created receptors allowing each metabolite concentration to be correlated with pathogen behavior. The development of the florescent strains used here provide initial maps of the reaction-diffusion dynamics existing in the rhizosphere, revealing significant differences in the signaling landscape of host and non-host plants before and after wounding, specifically highlighting networks that may inform rhizosphere organization.

A laminated energetic electrostatic analyzer for 0-5 keV charged particles.

A compact electrostatic energy bandpass filter based on a laminated analyzer design has been developed to measure charged particle fluxes at energies ranging from 0 to 5 keV. The sensor head has been successfully tested against a low energy magnetically filtered plasma source and an ion beam source capable of producing energetic ions in the range of 100-1250 eV. Additionally, the instrument has demonstrated the ability to accurately measure negative spacecraft frame charging using a low Earth orbit plasma simulator. The effects of the spacecraft frame charging on the measured energy distribution measurements and the impact regarding the derived charged particle density and temperature parameters are also examined.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass.

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

Advances in laser technology and fibre-optic delivery systems in lithotripsy.

The flashlamp-pumped, solid-state holmium:yttrium-aluminium-garnet (YAG) laser has been the laser of choice for use in ureteroscopic lithotripsy for the past 20 years. However, although the holmium laser works well on all stone compositions and is cost-effective, this technology still has several fundamental limitations. Newer laser technologies, including the frequency-doubled, double-pulse YAG (FREDDY), erbium:YAG, femtosecond, and thulium fibre lasers, have all been explored as potential alternatives to the holmium:YAG laser for lithotripsy. Each of these laser technologies is associated with technical advantages and disadvantages, and the search continues for the next generation of laser lithotripsy systems that can provide rapid, safe, and efficient stone ablation. New fibre-optic approaches for safer and more efficient delivery of the laser energy inside the urinary tract include the use of smaller-core fibres and fibres that are tapered, spherical, detachable or hollow steel, or have muzzle brake distal fibre-optic tips. These specialty fibres might provide advantages, including improved flexibility for maximal ureteroscope deflection, reduced cross section for increased saline irrigation rates through the working channel of the ureteroscope, reduced stone retropulsion for improved stone ablation efficiency, and reduced fibre degradation and burnback for longer fibre life.

Redox-mediated quorum sensing in plants.

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere's chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins' role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture.

Fiber optic muzzle brake tip for reducing fiber burnback and stone retropulsion during thulium fiber laser lithotripsy.

The experimental thulium fiber laser (TFL) is being explored as an alternative to the current clinical gold standard Holmium:YAG laser for lithotripsy. The near single-mode TFL beam allows coupling of higher power into smaller optical fibers than the multimode Holmium laser beam profile, without proximal fiber tip degradation. A smaller fiber is desirable because it provides more space in the ureteroscope working channel for increased saline irrigation rates and allows maximum ureteroscope deflection. However, distal fiber tip burnback increases as fiber diameter decreases. Previous studies utilizing hollow steel sheaths around recessed distal fiber tips reduced fiber burnback but increased stone retropulsion. A "fiber muzzle brake" was tested for reducing both fiber burnback and stone retropulsion by manipulating vapor bubble expansion. TFL lithotripsy studies were performed at 1908 nm, 35 mJ, 500 ?? ? s , and 300 Hz using a 100 - ? m -core fiber. The optimal stainless steel muzzle brake tip tested consisted of a 1-cm-long, 560 - ? m -outer-diameter, 360 - ? m -inner-diameter tube with a 275 - ? m -diameter through hole located 250 ?? ? m from the distal end. The fiber tip was recessed a distance of 500 ?? ? m . Stone phantom retropulsion, fiber tip burnback, and calcium oxalate stone ablation studies were performed ex vivo. Small stones with a mass of 40 ± 4 ?? mg and 4-mm-diameter were ablated over a 1.5-mm sieve in 25 ± 4 ?? s

Analysis of thulium fiber laser induced bubble dynamics for ablation of kidney stones.

The Thulium fiber laser (TFL) is being explored as an alternative to the Holmium : YAG laser for lithotripsy. TFL parameters differ in several fundamental ways from Holmium laser, including smaller fiber delivery, more strongly absorbed wavelength, low pulse energy/high pulse rate operation, and more uniform temporal pulse structure. High speed imaging of laser induced bubbles was performed at 105,000 frames per second and 10 µm spatial resolution to determine influence of these laser parameters on bubble formation and needle hydrophone data was also used to measure pressure transients. The TFL was operated at 1908 nm with pulse energies of 5-65 mJ, and pulse durations of 200-1000 µs, delivered through 105-µm-core and 270-µm-core silica optical fibers. Bubble dynamics using Holmium laser at a wavelength of 2100 nm with pulse energies of 200-1000 mJ and pulse duration of 350 µs was studied, for comparison. A single, 500 µs TFL pulse produced a bubble stream extending 1200 ± 90 µm and 1070 ± 50 µm from fiber tip, with maximum bubble widths averaging 650 ± 20 µm and 870 ± 40 µm (n = 4), for 105 µm and 270 µm fibers, respectively. These observations are consistent with previous studies which reported TFL ablation stallout at working distances beyond 1.0 mm. TFL bubble dimensions were four times smaller than for Holmium laser due to lower peak power and smaller fiber diameter used. The maximum pressure transients measured 0.6 bars at 35 mJ pulse energy for TFL and 7.5 bars at 600 mJ pulse energy for Holmium laser. These fundamental studies of bubble dynamics as a function of specific laser and fiber parameters may assist with optimization of the TFL parameters for safe and efficient lithotripsy in the clinic. Image of bubble formation during fiber optic delivery of Thulium fiber laser energy in saline (35 mJ, 500 µs).

A Rhizobium radiobacter Histidine Kinase Can Employ Both Boolean AND and OR Logic Gates to Initiate Pathogenesis.

The molecular logic gates that regulate gene circuits are necessarily intricate and highly regulated, particularly in the critical commitments necessary for pathogenesis. We now report simple AND and OR logic gates to be accessible within a single protein receptor. Pathogenesis by the bacterium Rhizobium radiobacter is mediated by a single histidine kinase, VirA, which processes multiple small molecule host signals (phenol and sugar). Mutagenesis analyses converged on a single signal integration node, and finer functional analyses revealed that a single residue could switch VirA from a functional AND logic gate to an OR gate where each of two signals activate independently. Host range preferences among natural strains of R. radiobacter correlate with these gate logic strategies. Although the precise mechanism for the signal integration node requires further analyses, long-range signal transmission through this histidine kinase can now be exploited for synthetic signaling circuits.

A Miniaturized, 1.9F Integrated Optical Fiber and Stone Basket for Use in Thulium Fiber Laser Lithotripsy.

The thulium fiber laser (TFL) is being explored as an alternative laser lithotripter to the standard holmium:yttrium-aluminum-garnet laser. The more uniform beam profile of the TFL enables higher power transmission through smaller fibers. In this study, a 100-µm core, 140-µm outer-diameter (OD) silica fiber with 5-mm length hollow steel tip was integrated with 1.3F (0.433-mm OD) nitinol wire basket to form a 1.9F (0.633-mm OD) device. TFL energy of 30 mJ, 500 µs pulse duration, and 500 Hz pulse rate was delivered to human uric acid stones, ex vivo. Stone ablation rates measured 1.5 ± 0.2 mg/s, comparable to 1.7 ± 0.3 mg/s using bare fiber tips separately with stone basket. With further development, this device may minimize stone retropulsion, allowing more efficient TFL lithotripsy at higher pulse rates. It may also provide increased flexibility, higher saline irrigation rates through the ureteroscope working channel, reduce fiber degradation compared with separate fiber and basket manipulation, and reduce laser-induced nitinol wire damage.

Collateral damage to the ureter and Nitinol stone baskets during thulium fiber laser lithotripsy.

BACKGROUND: The experimental Thulium fiber laser (TFL) is currently being studied as a potential alternative lithotripter to the clinical gold standard Holmium:YAG laser. Safety studies characterizing undesirable Holmium:YAG laser-induced damage to ureter tissue and stone baskets have been previously reported. Similarly, this study characterizes TFL induced ureter and stone basket damage. METHODS: A TFL beam with energy of 35 mJ per pulse, pulse duration of 500 µs, and variable pulse rates of 50-500 Hz, was delivered through 100-µm-core optical fibers, to either porcine ureter wall, in vitro, or a standard 1.9-Fr Nitinol stone basket wire. Ureter perforation times were measured and gross, histological, and optical coherence tomography images of the ablation area were acquired. Stone basket damage was graded as a function of pulse rate, number of pulses, and working distance. RESULTS: TFL operation at 150, 300, and 500 Hz produced mean ureter perforation times of 7.9, 3.8, and 1.8 seconds, respectively. Collateral damage widths averaged 510, 370, and 310 µm. Nitinol wire damage decreased with working distance and was non-existent at distances greater than 1.0 mm. In contact mode, 500 pulses delivered at pulse rates ≥300 Hz (≤1.5 seconds) were sufficient to cut Nitinol wires. CONCLUSIONS: The TFL, operated in low pulse energy and high pulse rate mode, may provide a greater safety margin than the standard Holmium:YAG laser for lithotripsy, as evidenced by longer TFL ureter perforation times and shorter non-contact working distances for stone basket damage than previously reported with Holmium:YAG laser.

Thulium fiber laser lithotripsy in an in vitro ureter model.

Using a validated in vitro ureter model for laser lithotripsy, the performance of an experimental thulium fiber laser (TFL) was studied and compared to the clinical gold standard holmium:YAG laser. The holmium laser (λ = 2120 nm) was operated with standard parameters of 600 mJ, 350 µs, 6 Hz, and 270-µm-core optical fiber. The TFL (λ=1908 nm) was operated with 35 mJ, 500 µs, 150 to 500 Hz, and a 100-µm-core fiber. Urinary stones (60% calcium oxalate monohydrate/40% calcium phosphate) of uniform mass and diameter (4 to 5 mm) were laser ablated with fibers through a flexible video-ureteroscope under saline irrigation with flow rates of 22.7 and 13.7 ml/ min for the TFL and holmium laser, respectively. The temperature 3 mm from the tube's center and 1 mm above the mesh sieve was measured by a thermocouple and recorded throughout each experiment for both lasers. Total laser and operation times were recorded once all stone fragments passed through a 1.5-mm sieve. The holmium laser time measured 167±41 s (n=12). TFL times measured 111±49, 39±11, and 23±4 s, for pulse rates of 150, 300, and 500 Hz, respectively (n=12 each). Mean peak saline irrigation temperatures reached 24±1°C for holmium, and 33±3°C, 33±7°C, and 39±6°C, for TFL at pulse rates of 150, 300, and 500 Hz, respectively. To avoid thermal buildup and provide a sufficient safety margin, TFL lithotripsy should be performed with pulse rates below 500 Hz and/or increased saline irrigation rates. The TFL rapidly fragmented kidney stones due in part to its high pulse rate, high power density, high average power, and observation of reduced stone retropulsion and may provide a clinical alternative to the conventional holmium laser for lithotripsy.

Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis.

Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.

Hollow steel tips for reducing distal fiber burn-back during thulium fiber laser lithotripsy.

The use of thulium fiber laser (TFL) as a potential alternative laser lithotripter to the clinical holmium:YAG laser is being studied. The TFL's Gaussian spatial beam profile provides efficient coupling of higher laser power into smaller core fibers without proximal fiber tip degradation. Smaller fiber diameters are more desirable, because they free up space in the single working channel of the ureteroscope for increased saline irrigation rates and allow maximum ureteroscope deflection. However, distal fiber tip degradation and "burn-back" increase as fiber diameter decreases due to both excessive temperatures and mechanical stress experienced during stone ablation. To eliminate fiber tip burn-back, the distal tip of a 150-µm core silica fiber was glued inside 1-cm-long steel tubing with fiber tip recessed 100, 250, 500, 1000, or 2000 µm inside the steel tubing to create the hollow-tip fiber. TFL pulse energy of 34 mJ with 500-µs pulse duration and 150-Hz pulse rate was delivered through the hollow-tip fibers in contact with human calcium oxalate monohydrate urinary stones during ex vivo studies. Significant fiber tip burn-back and degradation was observed for bare 150-µm core-diameter fibers. However, hollow steel tip fibers experienced minimal fiber burn-back without compromising stone ablation rates. A simple, robust, compact, and inexpensive hollow fiber tip design was characterized for minimizing distal fiber burn-back during the TFL lithotripsy. Although an increase in stone retropulsion was observed, potential integration of the hollow fiber tip into a stone basket may provide rapid stone vaporization, while minimizing retropulsion.

Pan1 is an intrinsically disordered protein with homotypic interactions.

The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp(312) and Trp(642) are each in an EH domain, Trp(957) is in the central region, and Trp(1280) is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.