Decellularized porcine peripheral nerve based injectable hydrogels as a Schwann cell carrier for injured spinal cord regeneration.

Objective.To develop a clinically relevant injectable hydrogel derived from decellularized porcine peripheral nerves and with mechanical properties comparable to native central nervous system (CNS) tissue to be used as a delivery vehicle for Schwann cell transplantation to treat spinal cord injury (SCI).Approach.Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. The decellularized nerves were delipidated using dichloromethane and ethanol solvent and then digested using pepsin enzyme to form injectable hydrogel formulations. Genipin was used as a crosslinker to enhance mechanical properties. The injectability, mechanical properties, and gelation kinetics of the hydrogels were further analyzed using rheology. Schwann cells encapsulated within the injectable hydrogel formulations were passed through a 25-gauge needle and cell viability was assessed using live/dead staining. The ability of the hydrogel to maintain Schwann cell viability against an inflammatory milieu was assessedin vitrousing inflamed astrocytes co-cultured with Schwann cells.Mainresults. The SDD method effectively removes cells and retains extracellular matrix in decellularized tissues. Using rheological studies, we found that delipidation of decellularized porcine peripheral nerves using dichloromethane and ethanol solvent improves gelation kinetics and mechanical strength of hydrogels. The delipidated and decellularized hydrogels crosslinked using genipin mimicked the mechanical strength of CNS tissue. The hydrogels were found to have shear thinning properties desirable for injectable formulations and they also maintained higher Schwann cell viability during injection compared to saline controls. Usingin vitroco-culture experiments, we found that the genipin-crosslinked hydrogels also protected Schwann cells from astrocyte-mediated inflammation.Significance. Injectable hydrogels developed using delipidated and decellularized porcine peripheral nerves are a potential clinically relevant solution to deliver Schwann cells, and possibly other therapeutic cells, at the SCI site by maintaining higher cellular viability and increasing therapeutic efficacy for SCI treatment.

Hypertonic saline- and detergent-accelerated EDTA-based decalcification better preserves mRNA of bones.

Ethylenediaminetetraacetic acid (EDTA), a classically used chelating agent of decalcification, maintains good morphological details, but its slow decalcification limits its wider applications. Many procedures have been reported to accelerate EDTA-based decalcification, involving temperature, concentration, sonication, agitation, vacuum, microwave, or combination. However, these procedures, concentrating on purely tissue-outside physical factors to increase the chemical diffusion, do not enable EDTA to exert its full capacity due to tissue intrinsic chemical resistances around the diffusion passage. The resistances, such as tissue inner lipids and electric charges, impede the penetration of EDTA. We hypothesized that delipidation and shielding electric charges would accelerate EDTA-based penetration and the subsequent decalcification. The hypothesis was verified by the observation of speedy penetration of EDTA with additives of detergents and hypertonic saline, testing on tissue-mimicking gels of collagen and adult mouse bones. Using a 26% EDTA mixture with the additives at 45°C, a conventional 7-day decalcification of adult mouse ankle joints could be completed within 24 h while the tissue morphological structure, antigenicity, enzymes, and DNA were well preserved, and mRNA better retained compared to using 15% EDTA at room temperature. The addition of hypertonic saline and detergents to EDTA decalcification is a simple, rapid, and inexpensive method that doesn't disrupt the current histological workflow. This method is equally or even more effective than the currently most used decalcification methods in preserving the morphological details of tissues. It can be highly beneficial for the related community.

Simple Rescue of Opaque Tissue Previously Cleared by iDISCO.

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time. Key features • This simple protocol rescues previously cleared tissue that has turned opaque. • The method does not cause detectable loss of immunofluorescence from previously stained samples. Graphical overview.

Nocardia seriolae cell wall lipids: An effective protective mechanism in resistance and virulence.

Piscine nocardiosis, caused by Nocardia seriolae, is a refractory granulomatous disease in South-East Asian aquaculture. This study investigates the virulence of nocardial lipids essential for pathogenesis among Actinomycetes. Petroleum ether (PE) was used to selectively delipidate two groups of N. seriolae, namely, live cell (LC) and killed cell (KC); resulting in delipidated live cell (DLC) and delipidated killed cell (DKC), respectively. Changes post-delipidation on genus characteristics, such as loss in acid-fast nature and resistance to lysozyme were observed. Transmission electron microscopy revealed notable changes in the lipid layer. Additionally, Lates calcarifer, Asian seabass intraperitoneally injected with LC and DLC had mortality rates of 90% and 50%, respectively, with the latter exhibiting a delay in mortality. Reverse-transcription quantitative PCR (RT-qPCR) analysis of host cytokines from the spleen and head kidney showed delipidation contributed to the induction of an immune response with increased transcriptional levels of interferon-γ (ifn-γ). Histopathological samples collected on day 7 post-inoculation displayed a varied granulomatous response between the treatment groups and scored for pathological changes. These findings affirm that the virulence of the lipids remains independent of the living state of the cell, significantly altering the immune and granulomatous responses in L. calcarifer to N. seriolae.

Selective delipidation of Mycobacterium bovis BCG retains antitumor efficacy against non-muscle invasive bladder cancer.

PURPOSE: Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment. MATERIALS AND METHODS: Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains. RESULTS: Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8+ T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8+ and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro. CONCLUSIONS: These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC.

Development of new tools to study membrane-anchored mammalian Atg8 proteins.

ABBREVIATIONS: A:C autophagic membrane:cytosol; ALS amyotrophic lateral sclerosis; ATG4 autophagy related 4; Atg8 autophagy related 8; BafA1 bafilomycin A1; BNIP3L/Nix BCL2 interacting protein 3 like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; EBSS Earle's balanced salt solution; GABARAP GABA type A receptor-associated protein; GST glutathione S transferase; HKO hexa knockout; Kd dissociation constant; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NLS nuclear localization signal/sequence; PE phosphatidylethanolamine; SpHfl1 Schizosaccharomyces pombeorganic solute transmembrane transporter; SQSTM1/p62 SQSTM1/p62; TARDBP/TDP-43 TAR DNA binding protein; TKO triple knockout.

Non-ionic hybrid detergents for protein delipidation.

Non-ionic detergents are important tools for the investigation of interactions between membrane proteins and lipid membranes. Recent studies led to the question as to whether the ability to capture protein-lipid interactions depends on the properties of detergents or their concentration in purification buffers. To address this question, we present the synthesis of an asymmetric, hybrid detergent that combines the head groups of detergents with opposing delipidating properties. We discuss detergent properties and protein purification outcomes to reveal whether the properties of detergent micelles or the detergent concentration in purification buffers drive membrane protein delipidation. We anticipate that our findings will enable the development of rationally design detergents for future applications in membrane protein research.

Roles of functional lipids in bacteriorhodopsin photocycle in various delipidated purple membranes.

Purple membrane (PM) is composed of several native lipids and the transmembrane protein bacteriorhodopsin (bR) in trimeric configuration. The delipidated PM (dPM) samples can be prepared by treating PM with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to partially remove native lipids while maintaining bR in the trimeric configuration. By correlating the photocycle kinetics of bR and the exact lipid compositions of the various dPM samples, one can reveal the roles of native PM lipids. However, it is challenging to compare the lipid compositions of the various dPM samples quantitatively. Here, we utilize the absorbances of extracted retinal at 382 nm to normalize the concentrations of the remaining lipids in each dPM sample, which were then quantified by mass spectrometry, allowing us to compare the lipid compositions of different samples in a quantitative manner. The corresponding photocycle kinetics of bR were probed by transient difference absorption spectroscopy. We found that the removal rate of the polar lipids follows the order of BPG ≈ GlyC < S-TGD-1 ≈ PG < PGP-Me ≈ PGS. Since BPG and GlyC have more nonpolar phytanyl groups than other lipids at the hydrophobic tail, causing a higher affinity with the hydrophobic surface of bR, the corresponding removal rates are slowest. In addition, as the reaction period of PM and CHAPS increases, the residual amounts of PGS and PGP-Me significantly decrease, in concomitance with the decelerated rates of the recovery of ground state and the decay of intermediate M, and the reduced transient population of intermediate O. PGS and PGP-Me are the lipids with the highest correlation to the photocycle activity among the six polar lipids of PM. From a practical viewpoint, combining optical spectroscopy and mass spectrometry appears a promising approach to simultaneously track the functions and the concomitant active components in a given biological system.

Sodium Cholate-Based Active Delipidation for Rapid and Efficient Clearing and Immunostaining of Deep Biological Samples.

Recent surges of optical clearing provided anatomical maps to understand structure-function relationships at organ scale. Detergent-mediated lipid removal enhances optical clearing and allows efficient penetration of antibodies inside tissues, and sodium dodecyl sulfate (SDS) is the most common choice for this purpose. SDS, however, forms large micelles and has a low critical micelle concentration (CMC). Theoretically, detergents that form smaller micelles and higher CMC should perform better but these have remained mostly unexplored. Here, SCARF, a sodium cholate (SC)-based active delipidation method, is developed for better clearing and immunolabeling of thick tissues or whole organs. It is found that SC has superior properties to SDS as a detergent but has serious problems; precipitation and browning. These limitations are overcome by using the ion-conductive film to confine SC while enabling high conductivity. SCARF renders orders of magnitude faster tissue transparency than the SDS-based method, while excellently preserving the endogenous fluorescence, and enables much efficient penetration of a range of antibodies, thus revealing structural details of various organs including sturdy post-mortem human brain tissues at the cellular resolution. Thus, SCARF represents a robust and superior alternative to the SDS-based clearing methods and is expected to facilitate the 3D morphological mapping of various organs.

Optimal delipidation solvent to secure extracellular matrix from human perirenal adipose tissue.

The objective of this study was to select the optimal delipidation solvent for preparation of human perirenal adipose tissue-derived extracellular matrix (ECM). Human perirenal adipose tissue can be obtained in large amounts during surgery, and it can be an alternative source of human ECM. Delipidation is an essential procedure for the ECM preparation, because lipid strongly inhibits regeneration of target tissue. Isopropanol has been widely used as a delipidation solvent for adipose tissue. However, because adipose tissue is mostly composed of nonpolar lipid, a nonpolar solvent might be more effective for delipidation. We evaluated the delipidation efficiency of acetone, chloroform, methanol, ether, ethanol, isopropanol, water, chloroform/methanol, ethanol/heptane, ether/methanol, hexane/ethanol, and butanol/methanol solvents for ECM extraction from human perirenal adipose tissue. Among them, acetone-treated adipose tissue showed the greatest delipidation efficiency (93.05%), significantly lower residual DNA content, and the greatest residual collagen concentration (42.49 ± 0.05 µg/g). In addition, acetone-treated tissue also had well-preserved ultrastructure with high porosity and significantly low in vitro cytotoxicity. These results suggested that acetone may be an optimal delipidation solvent for extraction of ECM from human perirenal adipose tissue.

Mechanosensitive channel gating by delipidation.

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-ß-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.

ATG4s: above and beyond the Atg8-family protein lipidation system.

The sole proteases of the macroautophagy/autophagy machinery, the ATG4s, contribute to autophagosome formation by cleaving Atg8-family protein members (LC3/GABARAPs) which enables Atg8-family protein lipidation and de-lipidation. Our recent work reveals that ATG4s can also promote phagophore growth independently of their protease activity and of Atg8-family proteins. ATG4s and their proximity partners including ARFIP2 and LRBA function to promote trafficking of ATG9A to mitochondria during PINK1-PRKN mitophagy. Through the development of a 3D electron microscopy framework utilizing FIB-SEM and artificial intelligence (termed AIVE: Artificial Intelligence-directed Voxel Extraction), we show that ATG4s promote ER-phagophore contacts during the lipid-transfer phase of autophagosome biogenesis, which requires ATG2B and ATG9A to support phagophore growth. We also discovered that ATG4s are not essential for removal of Atg8-family proteins from autolysosomes, but they can function as deubiquitinase-like enzymes to counteract the conjugation of Atg8-family proteins to other proteins, a process that we have termed ATG8ylation (also known as LC3ylation). These discoveries demonstrate the duality of the ATG4 family in driving autophagosome formation by functioning as both autophagy proteases and trafficking factors, while simultaneously raising questions about the putative roles of ATG8ylation in cell biology.

Lipid-free tuna muscle samples are suitable for total mercury analysis.

Tropical tunas are largely consumed worldwide, providing major nutritional benefits to humans, but also representing the main exposure to methylmercury, a potent neurotoxin that biomagnifies along food webs. The combination of ecological tracers (nitrogen and carbon stable isotopes, δ15N and δ13C) to mercury concentrations in tunas is scarce yet crucial to better characterize the influence of tuna foraging ecology on mercury exposure and bioaccumulation. Given the difficulties to get modern and historical tuna samples, analyses have to be done on available and unique samples. However, δ13C values are often analysed on lipid-free samples to avoid bias related to lipid content. While lipid extraction with non-polar solvents is known to have no effect on δ15N values, its impact on mercury concentrations is still unclear. We used white muscle tissues of three tropical tuna species to evaluate the efficiency and repeatability of different lipid extraction protocols commonly used in δ13C and δ15N analysis. Dichloromethane was more efficient than cyclohexane in extracting lipids in tuna muscle, while the automated method appeared more efficient but as repeatable as the manual method. Lipid extraction with dichloromethane had no effect on mercury concentrations. This may result from i) the affinity of methylmercury to proteins in tuna flesh, ii) the low lipid content in tropical tuna muscle samples, and iii) the non-polar nature of dichloromethane. Our study suggests that lipid-free samples, usually prepared for tropical tuna foraging ecology research, can be used equivalently to bulk samples to document in parallel mercury concentrations at a global scale.

Morphokinetic analysis of pronuclei using time-lapse cinematography in bovine zygotes.

The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.

Host Delipidation Mediated by Bacterial Effectors.

Protein lipidation, the covalent attachment of a lipid moiety to a target protein, plays a critical role in many cellular processes in eukaryotic cells. Bacterial pathogens secrete various effectors to subvert the host signaling pathway as a mechanism of microbial pathogenesis. An increasing number of effectors from diverse bacterial pathogens function as cysteine proteases to cause irreversible delipidation of host lipidated proteins. This in turn results in disruption of crucial lipidation-mediated host signal transduction, thereby enabling pathogen survival and replication. In this review, we discuss the role of the bacterial effectors in interactions with the host and highlight our knowledge of irreversible host delipidation, with a focus on the common concerted biochemical mechanisms of the bacterial effectors.

Optimised tissue clearing minimises distortion and destruction during tissue delipidation.

AIMS: A variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)-matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress. METHODS: We combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI-matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu-OptiClearing). Accu-OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens. RESULTS: Partial delipidation using Accu-OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu-OptiClearing compared to SDS. CONCLUSIONS: Combining the RI-matching solution OPTIClear with detergents, Accu-OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens.

The insufficiency of ATG4A in macroautophagy.

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

A solvent-free delipidation method for functional validation of lipases.

Extracting protein in its active form is critical for its functional characterization, and lipid removal is an essential step in the protein extraction process for further downstream applications. In the present study, we revisited the delipidation protocol and developed a rapid, solvent-free delipidation method using activated silica. The delipidated samples showed improved optical clarity and a significant reduction of endogenous lipids. The functional integrity of the lipases present in the delipidated sample was validated by in vitro enzyme assay using physiological substrate which includes neutral lipid as well as phospholipid. The accessibility of active site of the extracted enzymes was demonstrated by activity-based protein profiling (ABPP), a functional chemoproteomic approach. Detection of serine hydrolases using ABPP probe labeling was enhanced upon delipidation. Further, the total polyphenol content was significantly reduced, which helps to enhance the protein enrichment and small-molecule inhibitor screening by ABPP. Collectively, these results suggest that the present solvent-free delipidation approach is efficient and highly compatible with the functional characterization of the enzymes, particularly lipid hydrolases.

Synthesis and maintenance of lipid droplets are essential for mouse preimplantation embryonic development.

Lipid droplets (LDs), which are ubiquitous organelles consisting of a neutral lipid core coated with a phospholipid monolayer, play key roles in the regulation of cellular lipid metabolism. Although it is well known that mammalian oocytes and embryos contain LDs and that the amount of LDs varies among animal species, their physiological functions remain unclear. In this study, we have developed a method based on two-step centrifugation for efficient removal of almost all LDs from mouse MII oocytes (delipidation). We found that delipidated MII oocytes could be fertilized in vitro, and developed normally to the blastocyst stage even when the embryos were cultured in the absence of a fatty acid supply. LDs were newly synthesized and accumulated soon after delipidation, but chemical inhibition of long chain acyl-CoA synthetases (ACSLs) blocked this process, resulting in severe impairment of early embryonic development. Furthermore, we found that overabundance of LDs is detrimental to early embryonic development. Our findings demonstrate the importance of synthesis and maintenance of LDs, mediated in part by ACSL activity, during preimplantation embryonic development.