Direct Optical Patterning of Metal-Organic Frameworks via Photoacid-Induced Etching.

Metal-organic frameworks (MOFs) are a class of porous materials constructed from organic linkers and inorganic building blocks. Coordinative competition labilizes some MOFs under harsh chemical conditions because of their weak bonding. However, instability is not always a negative property of a material. In this study, we demonstrated the use of the acidic lability of MOFs for direct optical patterning. The controllable acid release from the photoacid generator at the exposed area causes bond cleavage between the linkers and metal ions/clusters, leading to solubility changes and pattern formation after development. This process avoids redundant steps and possible contamination in traditional photolithography, while maintaining the original properties of patterned MOFs. The preserved porosity and crystallinity promoted the development of MOFs for gas sensors and solid displays.

Crosslinking-induced patterning of MOFs by direct photo- and electron-beam lithography.

Metal-organic frameworks (MOFs) with diverse chemistry, structures, and properties have emerged as appealing materials for miniaturized solid-state devices. The incorporation of MOF films in these devices, such as the integrated microelectronics and nanophotonics, requires robust patterning methods. However, existing MOF patterning methods suffer from some combinations of limited material adaptability, compromised patterning resolution and scalability, and degraded properties. Here we report a universal, crosslinking-induced patterning approach for various MOFs, termed as CLIP-MOF. Via resist-free, direct photo- and electron-beam (e-beam) lithography, the ligand crosslinking chemistry leads to drastically reduced solubility of colloidal MOFs, permitting selective removal of unexposed MOF films with developer solvents. This enables scalable, micro-/nanoscale (≈70 nm resolution), and multimaterial patterning of MOFs on large-area, rigid or flexible substrates. Patterned MOF films preserve their crystallinity, porosity, and other properties tailored for targeted applications, such as diffractive gas sensors and electrochromic pixels. The combined features of CLIP-MOF create more possibilities in the system-level integration of MOFs in various electronic, photonic, and biomedical devices.

Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages.

Macrophages play an important role in maintaining tissue homeostasis, from regulating the inflammatory response to pathogens to resolving inflammation and aiding tissue repair. The surfactant protein A (SP-A) receptor SP-R210 (MYO18A) has been shown to affect basal and inflammatory macrophage states. Specifically, disruption of the longer splice isoform SP-R210L/MYO18Aα renders macrophages hyper-inflammatory, although the mechanism by which this occurs is not well understood. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of L isoform-deficient macrophages (SP-R210L(DN)) revealed basal and influenza-induced upregulation of genes associated with inflammatory pathways, such as TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockout of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) cells, with increased representation around genes relevant to inflammatory pathways. Additional ChIP-seq analysis of histone H3 methylation marks showed decreases in both repressive H3K9me3 and H3K27me3 marks with a commensurate increase in transcriptionally active (H3K4me3) histone marks in the L isoform deficient macrophages. Influenza A virus (IAV) infection, known to stimulate a wide array of anti-viral responses, caused a differential redistribution of PU.1 binding between proximal promoter and distal sites and decoupling from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. These finding suggest that the inflammatory differences seen in SP-R210L-deficient macrophages are a result of transcriptional differences that are mediated by epigenetic changes brought about by differential expression of the SP-R210 isoforms. This provides an avenue to explore how the signaling pathways downstream of the receptor and the ligands can modulate the macrophage inflammatory response.

Association of leptin and adiponectin levels with endometriosis: a systematic review and meta-analysis.

BACKGROUND: We aimed to summarize the available data regarding the levels of leptin and adiponectin and the key modulators of endometriosis compared to the controls. METHODS: The electronic databases such as MEDLINE, Embase, Scopus, Cochrane Library, and Web of Science were searched up to October 2020. The circulating and peritoneal levels of leptin and circulating levels of adiponectin were included. We used the Cochrane's Q test and the I2 statistic in this study. These tests' weighted mean difference (WMD) and 95% CIs were considered as the summary effect size. They were then pooled using a random-effects model with the DerSimonian-Laird method. RESULTS: Twenty eligible articles (or 25 studies) with 2645 participants (1362 women with endometriosis and 1283 controls) were included. Pooled results showed that women with endometriosis had significantly higher leptin levels (WMD = 4.45 mg/ml, 95%CI = 2.42-6.49, p < .01) and leptin/BMI ratio (WMD = 0.32 mg/ml, 95%CI = 0.23-0.42, p < .001) than the controls, whereas adiponectin levels (WMD = -0.24 mg/ml, 95%CI = -4.27 to -0.01, p = .038) were significantly lower. The pooled results also indicated significantly lower leptin levels in women with advanced-stage endometriosis (WMD = -8.07 mg/ml, 95%CI = -14.22 to -1.92, p = .01) than in the early stage. It was found, however, that there were no significant differences in adiponectin levels of women with advanced-stage endometriosis (WMD = -0.16 mg/ml, 95%CI = -0.64 to 0.32, p = .512) and the early-stage ones. CONCLUSION: We showed that leptin levels and leptin/BMI ratio were significantly higher in women with endometriosis than the controls. Nonetheless, patients with endometriosis had significantly lower levels of adiponectin than the controls.

An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy.

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.

Targeting the ESCRT-III component CHMP2A for noncanonical Caspase-8 activation on autophagosomal membranes.

Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia.

FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

ATG2 regulation of phagophore expansion at mitochondria-associated ER membranes.

The mechanism by which ATG2 (ATG2A and ATG2B in mammals) regulates autophagosome biogenesis remains largely unknown. In our recent study, we showed that ATG2A translocates to the mitochondria-associated ER membranes (MAM) to promote phagophore growth during nutrient starvation. Mechanistically, the mitochondrial translocase TOMM40 binds to a C-terminal domain of ATG2A, termed the MAM localization domain (MLD), and mediates its MAM translocation in a manner dependent on the TOMM receptor TOMM70. Moreover, ATG2A associates with ATG9A through its N-terminal domain and this interaction is required for phagophore expansion and efficient autophagic flux. These observations suggest that ATG2 operates a mechanism for phagophore expansion at the MAM through the TOMM40-TOMM70 complex and ATG9 during autophagy.

VPS37A directs ESCRT recruitment for phagophore closure.

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

TOM40 Targets Atg2 to Mitochondria-Associated ER Membranes for Phagophore Expansion.

During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.

An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure.

The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.

Analysis of Protein-Protein Interaction by Co-IP in Human Cells.

While there are various approaches available to analyze protein-protein interactions, coimmunoprecipitation (co-IP) remains one of the most classic and commonly used methods to discover novel protein interactions or to determine the physical association of proteins. The assay begins with the preparation of total cell or tissue lysate in an appropriate lysis buffer. Protein of interest in the lysate is captured using a specific antibody and precipitated along with its binding proteins using a resin. After a series of washes to remove nonbound proteins in the lysate, the resultant immune complexes are subjected to immunoblotting, in-gel protein staining, or mass spectrometry to determine the protein-protein interaction of interest. In this chapter, a standard IP/co-IP protocol is described and potential problems and troubleshooting are discussed.

Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis.

Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.

Endophilin B2 facilitates endosome maturation in response to growth factor stimulation, autophagy induction, and influenza A virus infection.

Endocytosis, and the subsequent trafficking of endosomes, requires dynamic physical alterations in membrane shape that are mediated in part by endophilin proteins. The endophilin B family of proteins contains an N-terminal Bin/amphiphysin/Rvs (N-BAR) domain that induces membrane curvature to regulate intracellular membrane dynamics. Whereas endophilin B1 (SH3GLB1/Bif-1) is known to be involved in a number of cellular processes, including apoptosis, autophagy, and endocytosis, the cellular function of endophilin B2 (SH3GLB2) is not well understood. In this study, we used genetic approaches that revealed that endophilin B2 is not required for embryonic development in vivo but that endophilin B2 deficiency impairs endosomal trafficking in vitro, as evidenced by suppressed endosome acidification, EGFR degradation, autophagic flux, and influenza A viral RNA nuclear entry and replication. Mechanistically, although the loss of endophilin B2 did not affect endocytic internalization and lysosomal function, endophilin B2 appeared to regulate the trafficking of endocytic vesicles and autophagosomes to late endosomes or lysosomes. Moreover, we also found that despite having an intracellular localization and tissue distribution similar to endophilin B1, endophilin B2 is dispensable for mitochondrial apoptosis. Taken together, our findings suggest that endophilin B2 positively regulates the endocytic pathway in response to growth factor signaling, autophagy induction, and viral entry.

The Bif-1-Dynamin 2 membrane fission machinery regulates Atg9-containing vesicle generation at the Rab11-positive reservoirs.

Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy.