Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI β; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Growth and maturation of the cerebrovasculature is a vital event in neocortical development however mechanisms that control cerebrovascular development remain poorly understood. Mutations in or deletions that include the FOXC1 gene are associated with congenital cerebrovascular anomalies and increased stroke risk in patients. Foxc1 mutant mice display severe cerebrovascular hemorrhage at late gestational ages. While these data demonstrate Foxc1 is required for cerebrovascular development, its broad expression in the brain vasculature combined with Foxc1 mutant's complex developmental defects have made it difficult to pinpoint its function(s). Using global and conditional Foxc1 mutants, we find 1) significant cerebrovascular growth defects precede cerebral hemorrhage and 2) expression of Foxc1 in neural crest-derived meninges and brain pericytes, though not endothelial cells, is required for normal cerebrovascular development. We provide evidence that reduced levels of meninges-derived retinoic acid (RA), caused by defects in meninges formation in Foxc1 mutants, is a major contributing factor to the cerebrovascular growth defects in Foxc1 mutants. We provide data that suggests that meninges-derived RA ensures adequate growth of the neocortical vasculature via regulating expression of WNT pathway proteins and neural progenitor derived-VEGF-A. Our findings offer the first evidence for a role of the meninges in brain vascular development and provide new insight into potential causes of cerebrovascular defects in patients with FOXC1 mutations.

Neural stem and precursor cell (NSPC) proliferation in the rodent adult hippocampus is essential to maintain stem cell populations and produce new neurons. Retinoic acid (RA) signaling is implicated in regulation of adult hippocampal neurogenesis, but its exact role in control of NSPC behavior has not been examined. We show RA signaling in all hippocampal NSPC subtypes and that inhibition of RA synthesis or signaling significantly decreases NSPC proliferation via abrogation of cell-cycle kinetics and cell-cycle regulators. RA signaling controls NSPC proliferation through hypoxia inducible factor-1α (HIF1α), where stabilization of HIF1α concurrent with disruption of RA signaling can prevent NSPC defects. These studies demonstrate a cell-autonomous role for RA signaling in hippocampal NSPCs that substantially broadens RA's function beyond its well-described role in neuronal differentiation.

Introduction Coronavirus disease 2019 (COVID-19) disease attributed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown associations with various fungal opportunistic infections such as mucormycosis, invasive candidiasis, and aspergillosis, which have contributed to the mortality of the disease. In India, the incidence of mucormycosis had risen rapidly during the second wave. There is ample literature demonstrating the role of iron in the pathogenesis of mucormycosis. The hyperferritinemia associated with COVID-19 may have played a significant role in promoting the invasion and extent of the fungus. Aims and objectives The study aimed to analyze the association between serum ferritin levels and the extent of involvement of mucormycosis in patients affected with COVID-19. Methodology A single-center cross-sectional study was conducted using retrospective hospital record data. G*Power statistical analysis software was used to compute the sample size of 62 (31+31). The radiological data were used to determine the extent of involvement. Results A statistically significant difference was seen in levels of serum ferritin (p = 0.008) between the radiologically judged two groups of the mild extent of invasion of mucormycosis (rhinosinusitis) and severe extent of invasion (rhino-orbital/cerebral mucormycosis), with a severe extent seen with the group having higher levels of serum ferritin. Severe extent of invasion was seen in 53.6% of patients with diabetes mellitus and 62.5% of patients with both diabetes and hypertension. Conclusion The hyperferritinemia not only presents as a marker of the systemic inflammatory process in COVID-19 but also indicates increased free iron, which thereby aids the growth and extent of involvement by the fungus (R hizopus oryzae). In individuals with diabetes and hypertension, the severity was greater. Controlling catastrophic outcomes in individuals with high serum ferritin levels necessitates extra caution.

An accurate and dimensionally stable trial denture base is required for a successful denture. The aim of this in vitro study was to assess the dimensional stability of a light-activated urethane dimethacrylate (UDMA) visible light cure (VLC) denture base with three fabrication techniques and different curing cycles. Forty-five VLC denture base samples were divided evenly into three groups. Group A used a conventional fabrication technique with a curing cycle of 5 min. Group B used a modified fabrication technique with two 4-min curing cycles. Group C used a multi-step fabrication technique with three curing cycles (4 min, plus 4 min, plus 2 min). The samples were sectioned and observed under a stereomicroscope to measure the discrepancy between the sample and the master cast. The mean dimensional discrepancy (mm) at the molar region at mid-palate, after 24 h in Group A, B and C was 0.790 mm, 0.741 mm and 0.379 mm, respectively; at the right ridge crest, it was 0.567, 0.408 and 0.185, while at the left ridge crest it was 0.475, 0.331 and 0.125, respectively. Statistical analysis showed significantly different dimensional discrepancies among the groups at all three sites; right ridge crest (F = 93.54, p < 0.001), left ridge crest (F = 105.96, p < 0.001) and mid-palate (F = 125.53, p < 0.001). Within the limitations of this laboratory study, it can be concluded that the denture base using a multi-step fabrication technique with three curing cycles provides better adaptation than the conventional technique. The significance of the study is that clinicians should consider performing denture base fabrication using a multi-step technique to enhance adaptation and hence the stability of the dentures for patients.

Silver nanoparticles were synthesized in the protein-polymer matrices of two different ratios to obtain a stringent control over the morphology. UV-visible spectrophotometry showed a single plasmon resonance peak at 416nm and 418nm respectively, confirming the formation of silver nanoparticles. X-ray diffractometry confirmed that the peaks matched with that of the reference silver. Both confocal microscopy and FEG-SEM confirmed the uniform morphology of the synthesized particles dependent on the template ratio. Doubling the protein-polymer concentration results in greater stability, more nucleation sites and hence restricted growth. Photoluminescence of the sample in the doubled matrix was found to be much greater at any given wavelength, meaning the flexibility and rigidity of interacting molecules affects the luminescence signal. The interaction in turn is dependent on the proximity of the proteins and polymer in the dispersion that forms a template and dictates the synthesis.

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer's disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD's cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell-derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).

Opioid overdose, dependence, and addiction are a major public health crisis. Patients with chronic kidney disease (CKD) are at high risk of opioid overdose, therefore novel methods that provide accurate prediction of renal clearance (CLr) and systemic disposition of opioids in CKD patients can facilitate the optimization of therapeutic regimens. The present study aimed to predict renal clearance and systemic disposition of morphine and its active metabolite morphine-6-glucuronide (M6G) in CKD patients using a vascularized human proximal tubule microphysiological system (VPT-MPS) coupled with a parent-metabolite full body physiologically-based pharmacokinetic (PBPK) model. The VPT-MPS, populated with a human umbilical vein endothelial cell (HUVEC) channel and an adjacent human primary proximal tubular epithelial cells (PTEC) channel, successfully demonstrated secretory transport of morphine and M6G from the HUVEC channel into the PTEC channel. The in vitro data generated by VPT-MPS were incorporated into a mechanistic kidney model and parent-metabolite full body PBPK model to predict CLr and systemic disposition of morphine and M6G, resulting in successful prediction of CLr and the plasma concentration-time profiles in both healthy subjects and CKD patients. A microphysiological system together with mathematical modeling successfully predicted renal clearance and systemic disposition of opioids in CKD patients and healthy subjects.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

Mitochondria play a prominent role in mechanosensory hair cell damage and death. Although hair cells are thought to be energetically demanding cells, how mitochondria respond to these demands and how this might relate to cell death is largely unexplored. Using genetically encoded indicators, we found that mitochondrial calcium flux and oxidation are regulated by mechanotransduction and demonstrate that hair cell activity has both acute and long-term consequences on mitochondrial function. We tested whether variation in mitochondrial activity reflected differences in the vulnerability of hair cells to the toxic drug neomycin. We observed that susceptibility did not correspond to the acute level of mitochondrial activity but rather to the cumulative history of that activity.

Recent genome-wide association studies have revealed genetic risk factors for Alzheimer's disease (AD) that are exclusively expressed in microglia within the brain. A proteomics approach identified moesin (MSN), a FERM (four-point-one ezrin radixin moesin) domain protein, and the receptor CD44 as hub proteins found within a co-expression module strongly linked to AD clinical and pathological traits as well as microglia. The FERM domain of MSN interacts with the phospholipid PIP2 and the cytoplasmic tails of receptors such as CD44. This study explored the feasibility of developing protein-protein interaction inhibitors that target the MSN-CD44 interaction. Structural and mutational analyses revealed that the FERM domain of MSN binds to CD44 by incorporating a beta strand within the F3 lobe. Phage-display studies identified an allosteric site located close to the PIP2 binding site in the FERM domain that affects CD44 binding within the F3 lobe. These findings support a model in which PIP2 binding to the FERM domain stimulates receptor tail binding through an allosteric mechanism that causes the F3 lobe to adopt an open conformation permissive for binding. High-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction, and one compound series was further optimized for biochemical activity, specificity, and solubility. The results suggest that the FERM domain holds potential as a drug development target. The small molecule preliminary leads generated from the study could serve as a foundation for additional medicinal chemistry effort with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM.

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity.