The Effects of Irradiation Time on Gelatin Methacrylate Hydrogels Used for Bone Tissue Engineering

Gelatin methacrylate (GelMA) hydrogels are a promising material for use in a variety of tissue engineering applications. Herein, we focused on identifying the optimal irradiation time necessary to photopolymerize GelMA hydrogels with visible blue light in a manner that did not adversely impact the biophysical properties of these cell-containing gels. We assessed the toxic effects of different irradiation times (3, 5, 10, 20 and 40 seconds) on BMMSCs encapsulated in a GelMA hydrogel using lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) as a photoinitiator. Both CCK-8 assays and Live-Dead staining were used to measure BMMSCs viability. We observed increasing compression strength as a function of increased irradiation time, although this corresponded to a reduction in swelling ratio and pore sizes. We ultimately found that when using LAP as a photoinitiator, the optimal irradiation time was 5–10 seconds, which was suitable for bone tissue engineering application. Ultimately we determined that a 5 second irradiation time was optimal for studies of encapsulated stem cells.

Realistic retinal modeling unravels the differential role of excitation and inhibition to starburst amacrine cells in direction selectivity

Retinal direction-selectivity originates in starburst amacrine cells (SACs), which display a centrifugal preference, responding with greater depolarization to a stimulus expanding from soma to dendrites than to a collapsing stimulus. Various mechanisms were hypothesized to underlie SAC centrifugal preference, but dissociating them is experimentally challenging and the mechanisms remain debatable. To address this issue, we developed the Retinal Stimulation Modeling Environment (RSME), a multifaceted data-driven retinal model that encompasses detailed neuronal morphology and biophysical properties, retina-tailored connectivity scheme and visual input. Using a genetic algorithm, we demonstrated that spatiotemporally diverse excitatory inputs–sustained in the proximal and transient in the distal processes–are sufficient to generate experimentally validated centrifugal preference in a single SAC. Reversing these input kinetics did not produce any centrifugal-preferring SAC. We then explored the contribution of SAC-SAC inhibitory connections in establishing the centrifugal preference. SAC inhibitory network enhanced the centrifugal preference, but failed to generate it in its absence. Embedding a direction selective ganglion cell (DSGC) in a SAC network showed that the known SAC-DSGC asymmetric connectivity by itself produces direction selectivity. Still, this selectivity is sharpened in a centrifugal-preferring SAC network. Finally, we use RSME to demonstrate the contribution of SAC-SAC inhibitory connections in mediating direction selectivity and recapitulate recent experimental findings. Thus, using RSME, we obtained a mechanistic understanding of SACs’ centrifugal preference and its contribution to direction selectivity.

Liposome assisted drug delivery-A review

Liposomes, sphere-formed vesicles consisting of one or greater phospholipid bilayers, had been first described within the mid-60s. Among numerous gifted new drug delivery systems, liposomes signify an advanced generation to supply active molecules to the site of action, and right now, numerous formulations are in clinical use. The application of liposomes to help drug shipping has already had a chief impact on many biomedical regions. They have been proven to be beneficial for stabilizing pharmaceuticals, overcoming boundaries to cellular and tissue uptake, and improving biodistribution of compounds to goal sites In vivo. This permits powerful delivery of encapsulated compounds to goal sites even as minimizing systemic toxicity. Liposomes present as an attractive transport gadget due to their wide physicochemical and biophysical properties which allow smooth manipulation to cope with exclusive shipping concerns. In this review, we will talk the advances in liposome assisted drug shipping, biological challenges, and present day medical and experimental use of liposomes for biomedical applications. The translational limitations of liposomal technology may also be provided.

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP′s many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterize the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observe an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a ′more mobile′ and a ′less mobile′ population: small particles in cells unable to make polyP are more likely to be ′mobile′ and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP. Our observations suggest that polyP limits cytoplasmic mobility and accessibility during nitrogen starvation, which may help to explain the pleiotropic phenotypes observed in the absence of polyP.

ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

Mycobacterium tuberculosis (Mtb) segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and Mtb intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens.

Parallel processing by distinct classes of principal neurons in the olfactory cortex

Understanding how distinct neuron types in a neural circuit process and propagate information is essential for understanding what the circuit does and how it does it. The olfactory (piriform, PCx) cortex contains two main types of principal neurons, semilunar (SL) and superficial pyramidal (PYR) cells. SLs and PYRs have distinct morphologies, local connectivity, biophysical properties, and downstream projection targets. Odor processing in PCx is thought to occur in two sequential stages. First, SLs receive and integrate olfactory bulb input and then PYRs receive, transform, and transmit SL input. To test this model, we recorded from populations of optogenetically identified SLs and PYRs in awake, head-fixed mice. Notably, silencing SLs did not alter PYR odor responses, and SLs and PYRs exhibited differences in odor tuning properties and response discriminability that were consistent with their distinct embeddings within a sensory-associative cortex. Our results therefore suggest that SLs and PYRs form parallel channels for differentially processing odor information in and through PCx.

Physiological and Pathophysiological Roles of Mitochondrial Na+-Ca2+ Exchanger, NCLX, in Hearts

It has been over 10 years since SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure–function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions—the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.

Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.