Thermoresponsive keratin-methylcellulose self-healing injectable hydrogel accelerating full-thickness wound healing by promoting rapid epithelialization.

Chronic wounds suffer from impaired healing due to microbial attack and poor vascular growth. Thermoresponsive hydrogels gained attention in wound dressing owing to their gelation at physiological temperature enabling them to take the shape of asymmetric wounds. The present study delineates the development of thermoresponsive hydrogel (MCK), from hair-derived keratin (K) and methylcellulose (MC) in the presence of sodium sulfate. The gelation temperature (Tg) of this hydrogel is in the range of 30 °C to 33 °C. Protein-polymer interaction leading to thermoreversible sol-gel transition involved in MCK blends has been analyzed and confirmed by FTIR, XRD, and thermal studies. Keratin, has introduced antioxidant properties to the hydrogel imparted cytocompatibility towards human dermal fibroblasts (HDFs) as evidenced by both MTT and live dead assays. In vitro wound healing assessment has been shown by enhanced migration of HDFs in the presence of MCK hydrogel compared to the control. Also, CAM assay and CD31 expression by the Wistar rat model has shown increased blood vessel branching after the implantation of MCK hydrogel. Further, in vivo study, demonstrated MCK efficacy of hydrogel in accelerating full-thickness wounds with minimal scarring in Wistar rats, re-epithelialization, and reinstatement of the epidermal-dermal junction thereby exhibiting clinical relevance for chronic wounds.

Natural Killer T cells and the invariant subset promote atherosclerosis: A meta-analysis.

AIMS: Natural Killer T (NKT) cells are reported to be both pro- and anti-atherosclerotic. With this meta-analysis, we evaluated the NKT population and their subsets in regulating the atherosclerotic disease in mice. MAIN METHODS: Eighteen pre-clinical (mice, n = 1276) and 6 clinical observational studies (humans, n = 116) met the eligibility criteria for inclusion. Random effects model was used and standard mean difference (SMD) was calculated for the cell counts and aortic lesion area. KEY FINDINGS: Lesion area decreased in the absence of whole NKT cell population (-1.33[95%CI, -2.14, -0.52]), and in the absence of only iNKT subset (-0.66[95%CI, -1.69, 0.37]). However, lesion area increased after over-expression/activation of iNKTs (1.40[95%CI, 0.28, 2.52]). Atherogenic diet (AD) or high fat diet (HFD) increased the number of NKT cells (2.51[95%CI, 1.42, 3.61]), whereas the iNKT cell numbers and iNKT cell-specific gene expression decreased in mice (-2.04[95%CI, -3.34, -0.75]) and atherosclerotic patients (-1.81[95 % CI, -2.89, -0.74]). SIGNIFICANCE: Here we show that, NKT and iNKT cells promote atherosclerosis. In general, NKT cell population increases with the progression of the plaque in mice and the numbers of iNKT cells reduce once the disease is established both in mice and humans.

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

Meta-analysis reveals inhibition of the inflammatory cytokine IL-6 affords limited protection post-myocardial ischemia/infarction.

Background: Proinflammatory cytokine cascades play crucial roles in the onset and progression of myocardial ischemia and infarction. Clinically, elevated serum levels of pro-inflammatory cytokine interleukin-6 is a poor prognostic indicator for future cardiac events and cardiac morbidity. Despite several reports, there is no clear evidence of cardiac benefits of inhibiting IL-6 in pre-clinical and clinical settings. Objective: To analyze the available data systematically and perform a meta-analysis to show the evidence of effects of IL-6 inhibition on cardiac remodeling and mortality in ischemic animal models. Methods: We used PICO framework and the quality of the studies was assessed using SYRCLE's risk of bias tool. Studies with interventions i.e., genetic deletion or pharmacological inhibition of IL-6/IL-6R were included for the meta-analysis. Systematic review was synthesized by including pre-clinical as well as randomized clinical trials involving myocardial infarction patients treated with IL-6 inhibitors. The effect size of the pooled data was determined using standard mean difference and 95% confidence intervals. Results: A total of 12 pre-clinical studies were included in the review for analysis. Most of the studies showed an unclear risk of bias as the selection and reporting criteria were poorly described. We observed high heterogeneity in the included studies due to the varying duration of myocardial infarction and the dosage of IL-6 antibodies used in the studies. Overall inhibition of IL-6 significantly increased area at risk [p = 0.001, SMD = 0.49 (95% CI: -0.36, 1.35)] and significantly reduced ejection fraction [p = 0.001, SMD = -0.19 (95% CI: -1.39, 1.01)] and end-diastolic diameter [p = 0.02, SMD = -0.25 (95% CI: -0.87, 0.36)] of left ventricle post-MI, but no effects on infarct size [p < 0.01, SMD = 0.00; 95% CI: -1.34, 0.58). In randomized clinical trials, the overall effect on C-reactive protein remains significantly unchanged on CRP levels (SMD = -0.38; 95% CI: -1.94, 0.55) post-treatment with IL-6R inhibitor tocilizumab. The meta-regression demonstrates a significant positive correlation (p = 0.058) between the increase in ischemic area and duration of ischemia post-surgery in the absence of IL-6. This meta-analysis indicates mixed effect of IL-6 inhibition on cardiac remodeling post-MI, particularly in protecting the myocardium viability from damaging acute inflammation but not significant on cardiac function of ischemic animal models. Conclusion: Despite the well-established pro-inflammatory nature of IL-6 in myocardial ischemia, our meta-analysis reports a limited contribution of IL-6 in the cardiac remodeling of hearts in animal models of myocardial ischemia. Moreover, genetically deleted IL-6 murine models produced contrasting results. Additional pre-clinical studies exploring the pharmacological inhibition of IL-6R are required to determine the beneficial effects of IL-6 inhibitors in regulating cardiac remodeling. The findings from IL-6R inhibition have better clinical relevance compared to genetically inhibited IL-6.

Inadvertent nucleotide sequence alterations during mutagenesis: highlighting the vulnerabilities in mouse transgenic technology.

In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as "junk DNA," have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.

Hematopoietic Stem Cell Transcription Factors in Cardiovascular Pathology.

Transcription factors as multifaceted modulators of gene expression that play a central role in cell proliferation, differentiation, lineage commitment, and disease progression. They interact among themselves and create complex spatiotemporal gene regulatory networks that modulate hematopoiesis, cardiogenesis, and conditional differentiation of hematopoietic stem cells into cells of cardiovascular lineage. Additionally, bone marrow-derived stem cells potentially contribute to the cardiovascular cell population and have shown potential as a therapeutic approach to treat cardiovascular diseases. However, the underlying regulatory mechanisms are currently debatable. This review focuses on some key transcription factors and associated epigenetic modifications that modulate the maintenance and differentiation of hematopoietic stem cells and cardiac progenitor cells. In addition to this, we aim to summarize different potential clinical therapeutic approaches in cardiac regeneration therapy and recent discoveries in stem cell-based transplantation.

Nanomachinery Organizing Release at Neuronal and Ribbon Synapses.

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.

Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.

Soluble immune checkpoint molecules: Serum markers for cancer diagnosis and prognosis.

BACKGROUND: With the recent advances in the understanding of the interaction of the immune system with developing tumor, it has become imperative to consider the immunological parameters for both cancer diagnosis and disease prognosis. Additionally, in the era of emerging immunotherapeutic strategies in cancer, it is very important to follow the treatment outcome and also to predict the correct immunotherapeutic strategy in individual patients. There being enormous heterogeneity among tumors at different sites or between primary and metastatic tumors in the same individual, or interpatient heterogeneity, it is very important to study the tumor-immune interaction in the tumor microenvironment and beyond. Importantly, molecular tools and markers identified for such studies must be suitable for monitoring in a noninvasive manner. RECENT FINDINGS: Recent studies have shown that the immune checkpoint molecules play a key role in the development and progression of tumors. In-depth studies of these molecules have led to the development of most of the cancer immunotherapeutic reagents that are currently either in clinical use or under different phases of clinical trials. Interestingly, many of these cell surface molecules undergo alternative splicing to produce soluble isoforms, which can be tracked in the serum of patients. CONCLUSIONS: Several studies demonstrate that the serum levels of these soluble isoforms could be used as noninvasive markers for cancer diagnosis and disease prognosis or to predict patient response to specific therapeutic strategies.

Vesicle sub-pool organization at inner hair cell ribbon synapses.

The afferent inner hair cell synapse harbors the synaptic ribbon, which ensures a constant vesicle supply. Synaptic vesicles (SVs) are arranged in morphologically discernable pools, linked via filaments to the ribbon or the presynaptic membrane. We propose that filaments play a major role in SV resupply and exocytosis at the ribbon. Using advanced electron microscopy, we demonstrate that SVs are organized in sub-pools defined by the filament number per vesicle and its connections. Upon stimulation, SVs increasingly linked to other vesicles and to the ribbon, whereas single-tethered SVs dominated at the membrane. Mutant mice for the hair cell protein otoferlin (pachanga, OtofPga/Pga ) are profoundly deaf with reduced sustained release, serving as a model to investigate the SV replenishment at IHCs. Upon stimulation, multiple-tethered and docked vesicles (rarely observed in wild-type) accumulated at OtofPga/Pga active zones due to an impairment downstream of docking. Conclusively, vesicles are organized in sub-pools at ribbon-type active zones by filaments to support vesicle supply, transport, and finally release.

The synaptic ribbon is critical for sound encoding at high rates and with temporal precision.

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation.

Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants.

The multi-C2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

Rab3-interacting molecules 2α and 2ß promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones.

Ca(2+) influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and ß (RIM2α and RIM2ß) in clustering voltage-gated CaV1.3 Ca(2+) channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2ß and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca(2+) imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca(2+) channels, resulting in reduced synaptic Ca(2+) influx. Using superresolution microscopy, we found that Ca(2+) channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca(2+) current, whereas the apparent Ca(2+) dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca(2+) influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2ß promote a large complement of synaptic Ca(2+) channels at IHC AZs and are required for normal hearing.

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.