Autonomous and directional flow of water and transport of particles across a subliming dynamic crystal surface.

Chemical and morphological traits of natural substrates that can propel and transport fluids over their surfaces have long provided inspiration for the engineering of artificial materials that can harvest and collect water from aerial humidity. Here we report that the gradual widening of parallel microchannels on a surface of a slowly subliming hexachlorobenzene crystal can promote the autonomous and bidirectional transduction of condensed aerial water. Driven by topology changes on the surface of the crystal and water exchange with the gas phase, droplets of condensed water migrate over the crystal. These droplets are also able to transport silver particles and other particulate matter, such as dust. The velocity of the particles was shown to be dependent on both the sublimation rate of the crystal and the relative humidity of its environment. This example of a sublimation-powered water flow demonstrates that topological surface changes accompanying crystal phase transitions can be harnessed to transport liquid and solid matter over surfaces.

Elastic Organic Crystals as Bioinspired Hair-Like Sensors.

One of the typical haptic elements are natural hairy structures that animals and plants rely on for feedback. Although these hair sensors are an admirable inspiration, the development of active flow sensing components having low elastic moduli and high aspect ratios remains a challenge. Here, we report a new sensing approach based on a flexible, thin and optically transmissive organic crystal of high aspect ratio, which is stamped with fluorescent dye for tracking. When subjected to gas flow and exposed to laser, the crystal bends due to exerted pressure and acts as an optical flow (hair) sensor with low detection limit (≈1.578 m s-1 ) and fast response time (≈2.70 s). The air-flow-induced crystal deformation and flow dynamics response are modelled by finite element analysis. Due to having a simple design and being lightweight and mechanically robust this prototypical crystal hair-like sensor opens prospects for a new class of sensing devices ranging from wearable electronics to aeronautics.

The ubiquitous microtubule-associated protein 4 (MAP4) controls organelle distribution by regulating the activity of the kinesin motor.

Regulation of organelle transport by molecular motors along the cytoskeletal microtubules is central to maintaining cellular functions. Here, we show that the ubiquitous tau-related microtubule-associated protein 4 (MAP4) can bias the bidirectional transport of organelles toward the microtubule minus-ends. This is concurrent with MAP4 phosphorylation, mediated by the kinase GSK3ß. We demonstrate that MAP4 achieves this bias by tethering the cargo to the microtubules, allowing it to impair the force generation of the plus-end motor kinesin-1. Consistent with this mechanism, MAP4 physically interacts with dynein and dynactin and, when phosphorylated, associates with the cargo-motor complex through its projection domain. Its phosphorylation coincides with the perinuclear accumulation of organelles, a phenotype that is rescued by abolishing the cargo-microtubule MAP4 tether or by the pharmacological inhibition of dynein, confirming the ability of kinesin to inch along, albeit inefficiently, in the presence of phosphorylated MAP4. These findings have broad biological significance because of the ubiquity of MAP4 and the involvement of GSK3ß in multiple diseases, more specifically in cancer, where the MAP4-dependent redistribution of organelles may be prevalent in cancer cells, as we demonstrate here for mitochondria in lung carcinoma epithelial cells.

Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis.

AMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

Viability assessment of human peripheral blood-derived stem cells after three methods of nebulization.

BACKGROUND AND OBJECTIVES: Drug delivery by nebulization has become a crucial strategy for treating different respiratory and lung diseases. Emerging evidence implicates stem cell therapy as a promising tool in treating such conditions, not only by alleviating the related symptoms but by improving the prognosis. However, delivery of human peripheral blood-derived stem cells (hPBSCs) to the respiratory airways remains an innovative approach yet to be realized. This study is an analytic, translational, and in vitro research to assess the viability and morphological changes of identified cell populations in hPBSCs cocktail derived from COVID-19 patients. METHODS AND RESULTS: Peripheral blood (PB) samples were obtained from patients enrolled in the SENTAD-COVID Study (ClinicalTrials.gov Reference: NCT04473170). hPBSCs cocktails (n=15) were provided by the Cells Processing Laboratory of Abu Dhabi Stem Cells Center, and were nebulized by three different methods of nebulization: compressor (jet), ultrasonic, and mesh. Our results reported that nucleated CD45dim cell count was significantly lower after the three nebulization methods, but nucleated CD45- cells show a significant decrease only after mesh nebulization. Mesh-nebulized samples had a significant reduction in viability of both CD45dim and CD45- cells. CONCLUSIONS: This study provides evidence that stem cells derived from PB of COVID-19 patients can be nebulized without substantial loss of cell viability, cell count, and morphological changes using the compressor nebulization. Therefore, we recommend compressor nebulizers as the preferable procedure for hPBSCs delivery to the respiratory airways in further clinical settings.

Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands.

The development of robust skeletal muscle models has been challenging due to the partial recapitulation of human physiology and architecture. Reliable and innovative 3D skeletal muscle models recently described offer an alternative that more accurately captures the in vivo environment but require an abundant cell source. Direct reprogramming or transdifferentiation has been considered as an alternative. Recent reports have provided evidence for significant improvements in the efficiency of derivation of human skeletal myotubes from human fibroblasts. Herein we aimed at improving the transdifferentiation process of human fibroblasts (tHFs), in addition to the differentiation of murine skeletal myoblasts (C2C12), and the differentiation of primary human skeletal myoblasts (HSkM). Differentiating or transdifferentiating cells were exposed to single or combinations of biological ligands, including Follistatin, GDF8, FGF2, GDF11, GDF15, hGH, TMSB4X, BMP4, BMP7, IL6, and TNF-α. These were selected for their critical roles in myogenesis and regeneration. C2C12 and tHFs displayed significant differentiation deficits when exposed to FGF2, BMP4, BMP7, and TNF-α, while proliferation was significantly enhanced by FGF2. When exposed to combinations of ligands, we observed consistent deficit differentiation when TNF-α was included. Finally, our direct reprogramming technique allowed for the assembly of elongated, cross-striated, and aligned tHFs within tissue-engineered 3D skeletal muscle constructs. In conclusion, we describe an efficient system to transdifferentiate human fibroblasts into myogenic cells and a platform for the generation of tissue-engineered constructs. Future directions will involve the evaluation of the functional characteristics of these engineered tissues.

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS-CoV-2 protease 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.

Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice.

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cellsLHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cellsLHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light-dark (LD) cycle. The cellsLHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cellsLHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cellsLHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cellsLHb→DRN and also show rapid photoentrainment to a new LD cycle.

Martensitic organic crystals as soft actuators.

Being capable of rapid and complete structure switching, the martensitic phase transitions in molecular crystals are thought to hold a tremendous potential as thermally driven organic actuators. However, the mechanical engineering parlance in the assessment of their performance is not immediately legible to the chemistry research community that starts to explore these materials, and the unavailability of performance indices has precluded molecular crystals from being considered in the device design process. Here, we demonstrate that an organic martensite, hexamethylbenzene, can be used to perform work that is comparable to that of most actuator classes. Millimeter-size single crystals of this material undergo a transition between two forms by uniaxial expansion at a rate of 6.36(2) mm s-1, exerting force in the range 10-100 mN. The force-to-weight ratio of the crystals is on the order of 104 and is superior to that of some living creatures. An actuator performance chart reveals that the performance of this material is close to that of nanomuscles, electrostatic actuators and voice coils, with a strain higher than that of electro/magnetostrictive actuators and ceramic piezoelectrics and stress higher than that of the electroactive polymers, MEMS devices, nanomuscles, voice coils, and some solenoids. Moreover, the crystals of this material are mechanically compliant and can be reversibly bent and shaped to fit the desired application. Altogether, the results point to the untapped potential of molecular crystals as rapid and efficient soft, organic actuators.

Spatial Photocontrol of the Optical Output from an Organic Crystal Waveguide.

The versatility in mechanical properties and the capability of optical waveguiding of molecular crystals have attracted research on the potential application of these materials in optomechanical transduction. Here, we demonstrate spatial photocontrol over the optical output from slender single crystals of an azo compound, 3',4'-dimethyl-4-(dimethylamino)azobenzene that can be used as a crystalline optical waveguide. The position of the free end of a single crystal can be controlled through reversible photoswitching between the trans and cis isomers at the irradiated crystal surface. The passive optical waveguiding capability of the crystal remains unaffected by its deformation induced by exposure to UV light. Moreover, the response time of the material by bending upon irradiation can be thermally regulated to control the positioning of the tip of the crystal. These single-crystal organic actuators with dual (optical and photomechanical) response deliver on the long sought for dynamic all-organic optical elements to be incorporated in microcircuits.

A filled organic crystal as a hybrid large-bandwidth optical waveguide.

A fluorescent hollow crystal of 9,10-dicyanoanthracene was filled with a perylene diimide derivative with complementary emissive features. The obtained crystal-in-crystal hybrid was used as a multiresponsive optical waveguide, which showed passive and active modes of light transduction and a bandwidth that extends from the visible to the infrared region.

Dual-Mode Light Transduction through a Plastically Bendable Organic Crystal as an Optical Waveguide.

An anthracene derivative, 9,10-dicyanoanthracene, crystallizes as fluorescent needle-like single crystals that can be readily plastically bent in two directions. Spatially resolved photoluminescence analysis revealed that this material has robust optoelectronic properties that are preserved upon extreme crystal deformation. The highly flexible crystals were successfully tested as efficient switchable optical waveguiding elements for both active and passive light transduction, and the mode of operation depends on the wavelength of the incident light. This prototypical dual-mode organic optical crystalline fiber brings mechanically compliant molecular organic crystals closer to applications as novel light-transducing media for wireless transfer of information in all-organic micro-optoelectronic devices.

Mesoporous γ-Iron Oxide Nanoparticles for Magnetically Triggered Release of Doxorubicin and Hyperthermia Treatment.

Mesoporous iron-oxide nanoparticles (mNPs) were prepared by using a modified nanocasting approach with mesoporous carbon as a hard template. mNPs were first loaded with doxorubicin (Dox), an anticancer drug, and then coated with the thermosensitive polymer Pluronic F108 to prevent the leakage of Dox molecules from the pores that would otherwise occur under physiological conditions. The Dox-loaded, Pluronic F108-coated system (Dox@F108-mNPs) was stable at room temperature and physiological pH and released its Dox cargo slowly under acidic conditions or in a sudden burst with magnetic heating. No significant toxicity was observed in vitro when Dox@F108-mNPs were incubated with noncancerous cells, a result consistent with the minimal internalization of the particles that occurs with normal cells. On the other hand, the drug-loaded particles significantly reduced the viability of cervical cancer cells (HeLa, IC50 =0.70 µm), wild-type ovarian cancer cells (A2780, IC50 =0.50 µm) and Dox-resistant ovarian cancer cells (A2780/AD, IC50 =0.53 µm). In addition, the treatment of HeLa cells with both Dox@F108-mNPs and subsequent alternating magnetic-field-induced hyperthermia was significantly more effective at reducing cell viability than either Dox or Dox@F108-mNP treatment alone. Thus, Dox@F108-mNPs constitute a novel soft/hard hybrid nanocarrier system that is highly stable under physiological conditions, temperature-responsive, and has chemo- and thermotherapeutic modes of action.

Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.

Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5' and 3'-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5' or 3' extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.