Membrane Protein Folding Research Articles

Expression, purification and folding of native like mitochondrial carrier proteins in lipid membranes

Mitochondrial Carrier Family proteins (MCFs) are located in the mitochondrial inner membrane and play essential roles in various cellular processes. Due to the relatively low abundance of many members of the family, in vitro structure and function determination of most MCFs require over-expression and purification of recombinant versions of these proteins. In this study, we report on a new method for overexpression of MCFs in Escherichia coli (E. coli) membranes, efficient purification of native-like proteins, and their reconstitution in mitochondrial inner membrane lipid mimics. cDNAs of Uncoupling Protein 4 (UCP4), Adenine Nucleotide Translocase (ANT) and Phosphate Translocase (PiT) were subcloned into the pET26b (+) expression vector such that fusion proteins with a short N-terminal pelB leader sequence and a six-histidine tag were produced to target the proteins toward the inner membrane of E. coli and facilitate affinity purification, respectively. Utilizing a modified autoinduction method, these proteins were overexpressed and extracted from the membrane of E. coli BL21 (DE3) and two modified strains, E. coli BL21 C43 (DE3) and E. coli BL21 Lobstr (DE3), in high yields. The proteins were then purified by immobilized metal affinity chromatography as monomers. Purity, identity, and concentration of the eluted monomers were determined by semi-native SDS-PAGE, Western blotting and mass spectrometry, and a modified Lowry assay, respectively. Cleavage of the pelB leader sequence from proteins was verified by mass spectrometric analysis. The purified proteins, surrounded by a shell of bacterial membrane lipids, were then reconstituted from the mild non-denaturing octyl glucoside (OG) detergent into phospholipid liposomes. Monomeric UCP4 spontaneously self-associated to form stable tetramers in lipid membranes, which is consistent with our previous studies. However, PiT and ANT remained dominantly monomeric in both detergent and liposome milieus, as detected by a combination of spectroscopic and electrophoretic methods. Native-like helical conformations of proteins were then confirmed by circular dichroism spectroscopy. Overall, this study demonstrates that targeting mitochondrial carrier family proteins to E. coli membranes provides an effective expression system for producing this family of proteins for biophysical studies.

Клинико-морфологическое обоснование двухэтапной хирургии осложненной катаракты и глаукомы

Aim: to provide a clinical and morphological basis for the efficacy of two-step surgery (phaco and trabeculectomy/TE) in patients with cataract and glaucoma. Patients and Methods: this study enrolled 87 patients with cataract and glaucoma. 18 patients (20.7%) were diagnosed with moderate glaucoma and 69 patients (79.3%) were diagnosed with advanced glaucoma. The intraocular pressure (IOP) was 28.4±1.4 mm Hg. Uncorrected visual acuity (UCVA) was 0.18±0.07 and best corrected visual acuity (BCVA) was 0.29±0.09. The first step was phaco with lens implantation. After 14 days, TE with scleral trephination was performed. Samples of the deep scleral flap, iris, and Tenon's capsule were collected during STE and sent for morphological examination to describe the nature of the tissue inflammatory response after cataract surgery. Results: 3 cases (3.4%) of malignant hypertension were detected after phaco. In the early postoperative period, ocular hypertension with corneal edema was diagnosed in 14 eyes (16.1%), Descemet's membrane folds in 16 eyes (18.4%), anterior chamber fibrin in 5 eyes (5.8%), and choroidal effusion in 1 eye (1.1%). After 14 days, IOP was 25.5±0.9 mm Hg, UCVA 0.5±0.06, and BCVA 0.56±0.05. After TE, hyphema and choroidal effusion were diagnosed in 29.9% (n=26) and 5.7% (n=5), respectively. One year after the two-step surgery, IOP was 18.5±1.1 mm Hg, UCVA 0.53±0.08, and BCVA 0.6±0.05. Two years after the two-step surgery, IOP was 18.8±0.7 mm Hg, UCVA 0.48±0.06, and BCVA 0.51±0.05. Morphological examination revealed signs of edema in all specimens and moderate congestion of Tenon's capsule. No cells typical of inflammation were detected. Conclusion: two-step surgery can be recommended for patients with cataract and uncontrolled glaucoma to control IOP and improve vision. KEYWORDS: glaucoma, cataract, phacoemulsification, trabeculectomy, intraocular lens, intraocular pressure, morphological examination. FOR CITATION: Ivachev E.A., Fedorova M.G. Clinical and morphological basis of two-step surgery for cataract and glaucoma. Russian Journal of Clinical Ophthalmology. 2024;24(1):19–24 (in Russ.). DOI: 10.32364/2311-7729-2024-24-1-4.

In Vitro Reconstruction of Bacterial β-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of β-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli(E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented β-barrel membrane proteins via intact β-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled β-barrel membrane proteins by demonstrating four representative β-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

In Vivo Disulfide-Bond Crosslinking to Study β-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded β-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of β-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of β-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between β-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.

Regulation of mitochondrial structure by the actin cytoskeleton.

Mitochondria are the powerhouse of the cell and play important roles in multiple cellular processes including cell metabolism, proliferation, and programmed cell death. Mitochondria are double-membrane organelles with the inner membrane folding inward to form cristae. Mitochondria networks undergo dynamic fission and fusion. Deregulation of mitochondrial structure has been linked to perturbed mitochondrial membrane potential and disrupted metabolism, as evidenced in tumorigenesis, neurodegenerative diseases, etc. Actin and its motors-myosins have long been known to generate mechanical forces and participate in short-distance cargo transport. Accumulating knowledge from biochemistry and live cell/electron microscope imaging has demonstrated the role of actin filaments in pre-constricting the mitochondria during fission. Recent studies have suggested the involvement of myosins in cristae maintenance and mitochondria quality control. Here, we review current findings and discuss future directions in the emerging fields of cytoskeletal regulation in cristae formation, mitochondrial dynamics, intracellular transport, and mitocytosis, with focus on the actin cytoskeleton and its motor proteins.

De Novo Granuloma of the Membranous Vocal Fold: A Marker of Occult Malignancy.

De novo occurrence of granuloma (granulation tissue) on the membranous vocal fold is not readily explained by usual causes of granuloma at the vocal process. We describe a series of patients. Case series. Single academic institution. Cases were identified over a 16-year period. All patients exhibited granulation tissue on pathology. Demographic details, presentation, treatment, histology, and clinical outcomes were recorded. Five patients (mean age: 74.0 ± 6.1 years, 40.0% male, 40.0% former smokers) underwent a biopsy. Persistent or recurrent granulation led to a second biopsy in 4 patients an average of 1423.5 days later, revealing a new diagnosis of squamous cell carcinoma (SCC) in situ in one and mild dysplasia in another. Further persistence or recurrence led to a third biopsy or excision an average of 302.3 days later in 3 patients, demonstrating SCC in situ in 1. An average of 2.5 biopsies were required with a mean time to SCC in situ diagnosis of 919.5 days from presentation. Two patients continued to demonstrate persistent granulation tissue on histology. The membranous vocal fold is an atypical location for granuloma and portends a risk of occult malignancy. The occurrence of de novo granuloma at this site should prompt close long-term clinical surveillance with a low threshold for biopsy. Consideration should be given to tissue collection in the operating room to adequately sample the lesion's base. Concern should persist even after a negative biopsy, and serial observation with repeat biopsy as needed should be pursued.

S-acylation: an orchestrator of the life cycle and function of membrane proteins.

S-acylation is a covalent post-translational modification of proteins with fatty acids, achieved by enzymatic attachment via a labile thioester bond. This modification allows for dynamic control of protein properties and functions in association with cell membranes. This lipid modification regulates a substantial portion of the human proteome and plays an increasingly recognized role throughout the lifespan of affected proteins. Recent technical advancements have propelled the S-acylation field into a 'molecular era', unveiling new insights into its mechanistic intricacies and far-reaching implications. With a striking increase in the number of studies on this modification, new concepts are indeed emerging on the roles of S-acylation in specific cell biology processes and features. After a brief overview of the enzymes involved in S-acylation, this viewpoint focuses on the importance of S-acylation in the homeostasis, function, and coordination of integral membrane proteins. In particular, we put forward the hypotheses that S-acylation is a gatekeeper of membrane protein folding and turnover and a regulator of the formation and dynamics of membrane contact sites.

Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.

Impact of cornea recovery training and certification on ophthalmology residents

BackgroundIn Asian countries, such as Taiwan, social taboos regarding organ and tissue donation decreases the prevalence of organ and tissue transplants. This also applies to cornea recovery, which is a skill that requires precision and practice to perform well. In Taiwan, to ensure the maintenance of high-quality corneas, a comprehensive training program and certified examination has been implemented. This study aims to investigate the impact of these programs and examinations on cornea recovery. MethodsResearchers evaluated the efficiency of the training and certified examination process by comparing the corneoscleral rim width, Descemet's membrane folds, endothelial layer stress lines, and endothelial cell density performed by ophthalmology residents in 2018 and 2019. ResultsAfter training and certification, the Descemet's membrane folds rate decreased from 14.3 % to 2.0 % and endothelial layer stress lines rate decreased from 22.5 % to 5.0 %. The endothelial cell density of donor grafts significantly improved from 2681.9 cells/mm2 to 2869.7 cells/mm2 (p < 0.001). ConclusionsThis study used objective data to evaluate cornea recovery quality after training and certification. The training and certified examination significantly improved the surgical skills of ophthalmology residents and could be applied in other tissue or organ recovery procedures to maintain and improve quality.

ASSESSMENT OF LABIAL FRENAL ATTACHMENT AND ITS ASSOCIATION WITH ORAL HYGIENE STATUS

Background: Maxillary labial frenum is a mucous membrane fold which shows variation in morphology, position. This anatomic structure presents with variety of orofacial problems leading to diastema, phonetic, periodontal, and orthodontic issues thus affecting growth and development. Hence it is mandatory to examine the frenal attachment during oral examination. With this the study was aimed to assess the prevalence of the various types of maxillary labial frenum attachment among the patients and its association with oral hygiene status. Methods: A total of 260 patients aged from 18 to 35years were clinically examined to evaluate the type of frenal attachment using Placek’s classification. Oral hygiene index (simplified) was recorded and data was analyzed statistically. The obtained data was statistically analyzed by using SPSS version 23.0 with descriptive and inferential statistics. Results: Most of the individuals 136 (52.3%) had gingival type of frenum followed by mucosal 92 (35.38%), papillary 21 (8.07%), and least with papillary penetrating 11(4.23%). There was statistically significance between frenum attachment and gender. The relationship of frenal attachment with oral hygiene status was found to be significant. Conclusions: In terms of maxillary labial frenum attachment type, gingival frenal type was the most predominant types among the study sample with a low incidence of papillary penetrating frenal attachment. The study showed strong association between type of frenal attachment and oral hygiene status.

MAXILLARY FRENECTOMY IN PEDIATRIC PATIENT USING ELECTROCAUTERY TECHNIQUE : REPORT OF 2 CASES

Background : The frenum is defined as a mocous membrane fold that attaches the lip and the cheek to the alveolar mucosa, the gingiva and the underlying periosteum. The unsual frenum need treated by frenectomy or by frenotomy procedure. Conventional technique of frenectomy carries the risks of surgery like bleeding and patient compliance with favorable healing. Electrocautery technique provide bloodless area and clear view of the operative field. Case : The present article is a report of two cases of frenectomy using electrocautery technique. Case management : Maxillary frenectomy of 2 patients was carried out using electrocautery technique after the area was anesthetized with 4% articaine. Continuous saline irrigation was given while using the electrocautery. Minimal bleeding was seen during the procedure. No sutures were placed after the treatment. One week recalled visit showed almost complete healing in both cases. No discomfort was complained by patinet during and after the treatment. Conclusion : Electrocautery is recommended for performing frenectomy due to its effectiveness and safety. The procedure causes light bleeding and does not have postoperative complications. The use of electrocautery in frenectomy provides a reduction in work time as well as greater comfort and safety for the patient dan dentist. Keywords : Frenectomy, Frenum, Pediatric Patient

Cx43-mediated hyphal folding counteracts phagosome integrity loss during fungal infection.

Phagolysosomes are crucial organelles during the elimination of pathogens by host cells. The maintenance of their membrane integrity is vital during stressful conditions, such as during Candida albicans infection. As the fungal hyphae grow, the phagolysosome membrane expands to ensure that the growing fungus remains entrapped. Additionally, actin structures surrounding the hyphae-containing phagosome were recently described to damage and constrain these pathogens inside the host vacuoles by inducing their folding. However, the molecular mechanism involved in the phagosome membrane adaptation during this extreme expansion process is still unclear. The main goal of this study was to unveil the interplay between phagosomal membrane integrity and folding capacity of C. albicans-infected macrophages. We show that components of the repair machinery are gradually recruited to the expanding phagolysosomal membrane and that their inhibition diminishes macrophage folding capacity. Through an analysis of an RNAseq data set of C. albicans-infected macrophages, we identified Cx43, a gap junction protein, as a putative player involved in the interplay between lysosomal homeostasis and actin-related processes. Our findings further reveal that Cx43 is recruited to expand phagosomes and potentiates the hyphal folding capacity of macrophages, promoting their survival. Additionally, we reveal that Cx43 can act as an anchor for complexes involved in Arp2-mediated actin nucleation during the assembly of actin rings around hyphae-containing phagosomes. Overall, this work brings new insights on the mechanisms by which macrophages cope with C. albicans infection ascribing to Cx43 a new noncanonical regulatory role in phagosome dynamics during pathogen phagocytosis. IMPORTANCE Invasive candidiasis is a life-threatening fungal infection that can become increasingly resistant to treatment. Thus, strategies to improve immune system efficiency, such as the macrophage response during the clearance of the fungal infection, are crucial to ameliorate the current therapies. Engulfed Candida albicans, one of the most common Candida species, is able to quickly transit from yeast-to-hypha form, which can elicit a phagosomal membrane injury and ultimately lead to macrophage death. Here, we extend the understanding of phagosome membrane homeostasis during the hypha expansion and folding process. We found that loss of phagosomal membrane integrity decreases the capacity of macrophages to fold the hyphae. Furthermore, through a bioinformatic analysis, we reveal a new window of opportunities to disclose the mechanisms underlying the hyphal constraining process. We identified Cx43 as a new weapon in the armamentarium to tackle infection by potentiating hyphal folding and promoting macrophage survival.

Protein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known ‘positive-outside’ rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. Invivo experiments using conditionally depleted E.coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. Invitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

Risk factors for postoperative vocal fold fibrosis following microlaryngeal surgery.

To analyze the risk factors for postoperative vocal fold fibrosis (PVF) in patients undergoing microlaryngeal surgery (MLS) for benign vocal fold lesions. Retrospective study. We retrospectively included patients who had undergone MLS for vocal polyps, nodules, mucus retention cysts, fibrous mass, or Reinke's edema. Data on the patients' clinicodemographic characteristics and intraoperative findings were obtained by reviewing their clinical records. PVF was defined by the presence of an adynamic segment of membranous vocal folds or a marked reduction in mucosal wave amplitude on post-MLS (6 weeks) videolaryngostroboscopy. The risk factors for PVF were analyzed through univariate and multivariate logistic regressions. This study included 89 patients, of whom 16 (18%) were given a diagnosis of PVF. A significantly increased incidence of PVF was noted in patients with fibrous mass (p < .01). The univariate analysis indicated that lesion attachment to the vocal ligament, prolonged surgical duration (>60 min), and symptom duration (>12 months) were significantly correlated with PVF (p < .05). The multivariate analysis confirmed that diagnosis of fibrous masses, lesion attachment to the vocal ligament and symptom duration are significant risk factors for PVF. PVF is more common in patients with fibrous masses. Lesions attachment to the vocal ligament and prolonged symptom duration appear to be other significant risk factors for PVF. 4.

Application of intraoperative optical coherence tomography in deep lamellar keratoplasty

Objective: To evaluate the clinical application value of intraoperative optical coherence tomography (iOCT) in deep anterior lamellar keratoplasty (DALK) using the big-bubble technique to bare Descemet's membrane. Methods: Retrospective case series. Clinical data of 92 patients (92 eyes) with monocular stromal corneal diseases who underwent big-bubble DALK in the Eye Hospital of Shandong First Medical University from January 2020 to August 2021 were collected. There were 53 males and 39 females. The average age was (53.2±16.0) years old. All patients underwent iOCT scanning to determine the location and depth of the injection needle after initial removal of the corneal lesion, to observe the integrity of the recipient bed, Descemet's membrane, after complete lesion removal, and to observe the adhesion between the corneal graft and the recipient bed and check folds on the recipient bed after suturing of the corneal graft. The intraoperative perforation of Descemet's membrane, postoperative thickness of the cornea and the recipient bed, visual acuity, and corneal astigmatism were recorded. Results: By iOCT, the thickness of the recipient bed was found to be about 1/2 of the corneal thickness and relatively uniform in all directions in 62 eyes (67.4%), so the sterile air was injected from the center of the recipient bed to separate it from the stromal layer. In 30 eyes (32.6%) with an uneven thickness of the recipient bed, the sterile air was injected from the paracentral area of the recipient bed. Under the guidance of iOCT scanning, 89 eyes (96.7%) did not experience any perforation of Descemet's membrane during surgery. The Descemet's membrane folds in the central 5-mm area of the recipient bed was observed and flattened in 20 eyes with the assistance of iOCT scanning. The postoperative corneal thickness was (578.95±108.26) μm, and the recipient bed thickness was (36.06±23.11) μm. The best corrected visual acuity of all patients at 6 months after surgery was 0.57±0.25 logMAR, which was significantly better than that before surgery (1.61±1.27 logMAR; P<0.001). The average corneal astigmatism at 6 months after surgery was (2.72±2.44) diopters. Conclusions: The application of iOCT scanning in DALK surgery assisted by the big-bubble method can provide safe guidance for surgeons to adopt correct surgical procedures, decrease the risk of Descemet's membrane perforation, reduce the recipient bed folds, and facilitate corneal interlayer adhesion, thereby improving the visual prognosis.

A Case of Hypertensive Anterior Uveitis with Endotheliitis of the Cornea Following COVID-19 Pneumonia

Purpose: We report a patient diagnosed with COVID-19-associated hypertensive anterior uveitis and endotheliitis of the cornea.Case Summary: A 71-year-old male visited our hospital with decreased visual acuity, ocular pain, eyelid swelling of in his right eye, and headache for 8 days. He had been diagnosed with COVID-19 pneumonia 10 days before the visit. The best-corrected visual acuity was 0.04 in his right eye and 0.15 in his left eye. Severe conjunctival injection, moderate corneal edema with Descemet membrane folding, diffuse keratic precipitates and mild anterior chamber reaction were observed in the right eye, which were not present in the previous examination. Pupils in both eyes were moderately dilated and slow to react in response to direct light. The density of endothelial cells in the right eye was 711 cells/mm&lt;sup&gt;2&lt;/sup&gt; and an owl’s eye morphology of endothelial cells was observed via specular microscopy. During follow up, elevated intraocular pressure (IOP) in the right eye was noted. Polymerase chain reaction (PCR) examination of the aqueous humor for cytomegalovirus was negative. After 2 weeks of applying prednisolone eye drops and anti-glaucoma eye drops, corneal edema decreased, and his vision improved to 0.5. However, the density of the corneal endothelium remained low.Conclusions: Although there have been worldwide case reports of conjunctivitis, keratitis, or uveitis after COVID-19 infection, this study presents the first reported case of COVID-19 associated hypertensive anterior uveitis and endotheliitis of the cornea.

Nonlinear wrap-folding of membranes with predefined creases and seams

The investigation of folding processes of aerospace membrane structures holds significant importance due to the limited volume of launch vehicles. This paper presents a simulation system for membrane wrap-folding with predefined creases and seams. To transform non-rigid foldable configurations into rigid foldable ones, the methods of adding creases and cutting strategies are introduced. The formulation of constraint equations for the hinged rod system model enables the analysis of the rigid folding motion. A two-stage folding strategy is also implemented for fully folding, consisting of an initial rigid folding stage followed by an additional displacement folding stage. By incorporating the crease strategy and employing a two-stage driving mode, the stress levels throughout the folding process are minimized. Furthermore, accounts for the presence of seam areas, placing the seam at the crease creates wrinkles smaller when folded. The findings contribute to a deeper understanding of membrane folding behavior, leading to advancements in their design and application.

Visualizing Formation and Dynamics of a Three-Dimensional Sponge-like Network of a Coacervate in Real Time.

Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and formation mechanism of coacervates remain elusive. Herein, we demonstrate real-time confocal observation of a three-dimensional sponge-like network in a dipeptide-based coacervate. In situ generation of the dipeptide allowed us to capture the emergence of the sponge-like network via unprecedented membrane folding of vesicle-shaped intermediates. We also visualized dynamic fluctuation of the network, including reversible engagement/disengagement of cross-links and a stochastic network kissing event. Photoinduced transient formation of a multiphase coacervate was achieved with a thermally responsive phase transition. Our findings expand the fundamental understanding of synthetic coacervates and provide opportunities to manipulate their physicochemical properties by engineering the inner network for potential applications in development of artificial cells and life-like material fabrication.

Subtotal Submucosal Arytenoidectomy with Lateralization Sutures (SMALS) Covered by a Mucosal Flap.

Glottic airway stenosis (GAWS) may result from bilateral paralysis (BVFP) or posterior glottic stenosis (PGS). Since the glottis is the principal airway sphincter, surgeons shift on the balance between airway, aspirations, and voice. We aim to describe our surgical technique and outcome of the SMALS procedure for GAWS correction. A retrospective cohort of patients who underwent SMALS for PGS between 2018 and 2021. SMALS involves: endoscopic submucosal subtotal arytenoidectmy (preserving medial mucosal flap) and lateralization sutures. The sutures lateralize the mucosal flap to cover the arytenoidectomy bed without lateralization of the membranous vocal fold; expanding the posterior glottis, while preserving a relatively good voice. Covering the arytenoidectomy bed enhances healing. Medical and surgical data, airway, voice, and swallowing symptoms were collected. Relative glottic opening area (RGOA) and relative glottic insufficiency area (RGIA) were calculated. Eleven PGS patients who underwent 15 SMALS were included (4 bilateral), all patients had post-intubation PGS, 1 patient also had prior radiation to the larynx. All patients were tracheostomy-dependent. There were no major complications. No granulation or retracting scar was observed at follow-up. None had a persistent voice or swallowing disability. Successful outcome (decannulation) was achieved in 8 (73%); RGOA increased in all (Δ = 0.37; p = 0.003), while RGIA remained relatively stable (Δ = 0.02; p = 0.055). SMALS is a safe and effective, novel modification of the classic arytenoidectomy, for GAWS correction that can be easily applied and may expand the airway without significant glottic insufficiency symptoms. 4 Laryngoscope, 134:353-360, 2024.