Recombinant manufacturing of multispecies biolubricants.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O -glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Size-selective adhesion of calcium oxalate monohydrate crystals to lipid membranes.

The retention of calcium oxalate monohydrate (COM) crystals on cell membranes is pivotal in kidney stone formation. However, the mechanisms underlying COM attachment to neutral lipid membranes remain unclear. In this study, we demonstrate that COM exhibits size-selective adhesion to fluid lipid membranes composed of lipids with distinct sizes. Specifically, the (100) facet of COM induces the formation of new domains and establishes strong adhesion in the 18:1 (Δ9-Cis) PC (DOPC) membrane, while the (010) facet induces domains with strong adhesion in the 16:0-14:0 PC membrane. This selectivity is linked to the compatibility of the area per lipid in DOPC with the unit cell area of the (100) facet and the area per lipid in 16:0-14:0 PC with the (010) facet. Our Raman spectroscopic analyses reveal that the lipid acyl chains within these induced domains exhibit a higher degree of ordering compared to the typical fluid state of the membrane. This ordered structural alignment, combined with the lateral size-matching effect, suggests the potential formation of molecular arrays within the lipid bilayer that are in harmony with the lattice dimension of COM. To elucidate the strong adhesion between calcium oxalate and the phospholipid head group in the absence of a direct molecular structural correspondence, we propose that crystal water associated with COM can form hydrogen bonds with the phospholipid head group. Using structure visualization software, we demonstrate the feasibility of such hydrogen bonding networks. The formation of this network could serve to stabilize and enhance the attachment of COM to the lipid membrane. This mediation by water molecules offers a plausible explanation for the pronounced affinity at the interface.

3D Foaming Printing Biomimetic Hierarchically Macro-Micronanoporous Hydrogels for Enhancing Cell Growth and Proliferation.

Hydrogel, recognized as a promising biomaterial for tissue engineering, possesses notable characteristics, including high water uptake, an interconnected porous structure, and excellent permeability. However, the intricate task of fabricating a hierarchically macro-micronanoporous structure, essential for providing adequate space for nutrient diffusion and cell growth within hydrogels, remains a formidable challenge. In response to these challenges, this study introduces a sustainable and straightforward three-dimensional (3D) foaming printing strategy to produce hierarchically macro-micronanoporous hydrogels (HPHs) without the utilization of porogens and post-etching process. This method entails the controlled generation of air bubbles within the hydrogels through the application of optimal mechanical stirring rates. Subsequent ultraviolet (UV) cross-linking serves to effectively stabilize the macropores within the HPHs. The resulting hierarchically macro-micronanoporous structures demonstrate a substantial improvement in the viability, adhesion, and proliferation of human umbilical vein endothelial cells (HUVECs) when incubated with the hydrogels. These findings present a significant advancement in the fabrication of hierarchically macro-micronanoporous hydrogels, with potential applications in the fields of tissue engineering and organoid development.

De Garengeot Hernia, an acute appendicitis in the right femoral hernia canal, and successful management with transabdominal closure and appendectomy: a case Report.

Incarceration of the appendix within a femoral hernia is a rare condition of abdominal wall hernia about 0.1 to 0.5% in reported femoral hernia [1]. We report a case of a 56-year-old female whose appendix was trapped in the right femoral canal. There are few reports in the literature on entrapment of the appendix within a femoral hernia. The management of this condition includes antibiotics, drainage appendectomy, hernioplasty and mesh repair.

Recombinant Production of Glycoengineered Mucins in HEK293-F Cells.

Recombinant mucins are attractive polymeric building blocks for new biomaterials, biolubricants, and therapeutics. Advances in glycoengineered host cell systems now enable the recombinant production of mucins with tailored O-glycan side chains, offering new opportunities to tune the functionality of mucins and investigate the biology of specific O-glycan structures. Here, we provide a protocol for the scalable production of glycoengineered mucins and mucin-like glycoproteins in suspension-adapted HEK293-F cells. The protocol includes the preparation of engineered cell lines with homozygous knockout (KO) of glycosyltransferases using CRISPR/Cas9 and homology-directed repair (HDR) templates designed for efficient screening of clones. Strategies are provided for the stable introduction of mucin expression cassettes into the HEK293-F genome and the subsequent isolation of high-expressing cell populations. The high-titer production of recombinant mucins in conventional shaker flasks is described as an example production strategy using these cell lines.

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O -glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O -glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O -glycosylation in the first larval stage and identify O -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans , we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

Azo-Cyanamide Bridged Dinuclear Iron Complexes Exhibiting no Electronic Coupling but Moderate Magnetic Coupling between the two Iron Centers.

To investigate the effect of long-distance organic ligand on electronic coupling between metallic atoms, the mononuclear and dinuclear complexes [Cp(dppe)Fe(apc)] (1), [{Cp(dppe)Fe}2(µ-adpc)] (2), [{CpMe5(dppe)Fe}2(µ-adpc) (3) and their oxidized complexes [Cp(dppe)Fe(apc)][PF6] (1[PF6]), [{Cp(dppe)Fe}2(µ-adpc)][PF6] (2[PF6]2), [{CpMe5(dppe)Fe}2(µ-adpc)][PF6]2 (3[PF6]2) (Cp=1,3-cyclopentadiene, CpMe5=1,2,3,4,5-pentamethylcyclopentadiene, dppe=1,2-bis(diphenylphosphino)ethane), apc-=4-azo(phenylcyanamido)benzene and adpc2-=4,4'-azodi(phenylcyanamido)) were synthesized and characterized by cyclic voltammetry, UV-vis, single-crystal X-ray diffraction and Mössbauer spectra. Electrochemical measurements showed no electronic coupling between the two terminal Fe units, However, the investigation results of the magnetic properties of the two-electron oxidized complexes indicate the presence of moderate antiferromagnetic coupling across 18 Šdistance.

Lysine-372-dependent SUMOylation inhibits the enzymatic activity of glutamine synthases.

Glutamine synthetase (GS) is a crucial enzyme involved in de novo synthesis of glutamine and participates in several biological processes, including nitrogen metabolism, nucleotide synthesis, and amino acid synthesis. Post-translational modification makes GS more adaptable to the needs of cells, and acetylation modification of GS at double sites has attracted considerable attention. Despite very intensive research, how SUMOylation affects GS activity at a molecular level remains unclear. Here, we report that previously undiscovered GS SUMOylation which is deficient mutant K372R of GS exhibits more bluntness under glutamine starvation. Mechanistically, glutamine deprivation triggers the GS SUMOylation, and this SUMOylation impaired the protein stability of GS, within a concomitant decrease in enzymatic activity. In addition, we identified SAE1, Ubc9, and PIAS1 as the assembly enzymes of GS SUMOylation respectively. Furthermore, Senp1/2 functions as a SUMO-specific protease to reverse the SUMOylation of GS. This study provides the first evidence that SUMOylation serves as a regulatory mechanism for determining the GS enzymatic activity, contributing to understanding the GS regulation roles in various cellular and pathophysiological processes.

Evaluation of Risk Prediction with Hierarchical Data: Dependency Adjusted Confidence Intervals for the AUC.

The area under the true ROC curve (AUC) is routinely used to determine how strongly a given model discriminates between the levels of a binary outcome. Standard inference with the AUC requires that outcomes be independent of each other. To overcome this limitation, a method was developed for the estimation of the variance of the AUC in the setting of two-level hierarchical data using probit-transformed prediction scores generated from generalized estimating equation models, thereby allowing for the application of inferential methods. This manuscript presents an extension of this approach so that inference for the AUC may be performed in a three-level hierarchical data setting (e.g., eyes nested within persons and persons nested within families). A method that accounts for the effect of tied prediction scores on inference is also described. The performance of 95% confidence intervals around the AUC was assessed through the simulation of three-level clustered data in multiple settings, including ones with tied data and variable cluster sizes. Across all settings, the actual 95% confidence interval coverage varied from 0.943 to 0.958, and the ratio of the theoretical variance to the empirical variance of the AUC varied from 0.920 to 1.013. The results are better than those from existing methods. Two examples of applying the proposed methodology are presented.

ZNF205 positively regulates RLR antiviral signaling by targeting RIG-I.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic viral RNA receptor. Upon viral infection, the protein recognizes and then recruits adapter protein mitochondrial antiviral signaling (MAVS) protein, initiating the production of interferons and proinflammatory cytokines to establish an antiviral state. In the present study, we identify zinc finger protein 205 (ZNF205) which associates with RIG-I and promotes the Sendai virus (SeV)-induced antiviral innate immune response. Overexpression of ZNF205 facilitates interferon-beta (IFN-ß) introduction, whereas ZNF205 deficiency restricts its introduction. Mechanistically, the C-terminal zinc finger domain of ZNF205 interacts with the N-terminal tandem caspase recruitment domains (CARDs) of RIG-I; this interaction markedly promotes K63 ubiquitin-linked polyubiquitination of RIG-I, which is crucial for RIG-I activation. Thus, our results demonstrate that ZNF205 is a positive regulator of the RIG-I-mediated innate antiviral immune signaling pathway.

Endemic melioidosis in central Taiwan-A longitudinal case cohort study.

Background: Melioidosis is a systemic and suppurative disease endemic in the Southeast Asia. In Taiwan, most cases are reported in the southern region and no relevant profiles have been reported in central region. In this study, we performed the epidemiologic and clinical analyses from the melioidosis cases in central Taiwan. Methods: The demographic, clinical, laboratory, radiologic, and outcome profiles were collected retrospectively and analyzed from patients whom Burkhoderia pseudomallei was isolated from clinical specimens during the 12-year study period (2011-2022). Results: Totally 11 melioidosis cases (10 males and 1 female) were diagnosed, among them only 2 (18.2%) cases lived in suburban areas. Seven (63.6%) cases were diagnosed during 2019-2020, and diabetes mellitus was the most relevant comorbidity (5, 45.4%). All cases presented with fever at arrival, but only 4 (36.4%) and 2 (18.2%) cases presented with dyspnea and shock, respectively. Pneumonitis and extrapulmonary involvement were found in 5 cases (45.4%) each. Appropriate empiric and targeted antibiotic treatments were found in 4 (36.4%) and 10 (91.0%) case, respectively. Two cases (18.2%) succumbed to infection despite appropriate treatment including targeted antibiotics. Conclusion: Melioidosis has become endemic in central Taiwan. Septic patients who present with suppurative or undetermined foci and have unsatisfied responses to standard treatment should arouse clinicians to take melioidosis into consideration.

Trophic niche and potential carbon source of three reef-associated fishes of Zhongjieshan Islands.

To clarify the trophic relationship of important rock fishes, we analyzed trophic niche of three typical rockfish species (Oplegnathus fasciatus, Sebastiscus marmoratus and Conger myriaster) in the Zhongjieshan Islands in summer 2020, based on the carbon and nitrogen stable isotope techniques. We calculated the contributions of major carbon sources [macroalgae, phytoplankton, suspended particulate organic matter (POM) and substrate organic matter (SOM)]. The results showed that: 1) the δ13C values of the three species ranged from -21.44‰ to -15.21‰, with an average value of (-16.85±1.12)‰, while the δ15N values ranged from 8.32‰ to 10.96‰, with an average value of (9.69±0.66)‰. There were significant differences in carbon and nitrogen stable isotopes among the three species. 2) There was small niche overlap between O. fasciatus and S. marmoratus, indicating that the interspecific competition was not intense. There was no overlap between C. myriaster and the first two, indicating feeding differentiation. 3) The total ecotone area, corrected core ecotone area, and food source diversity of C. myriaster were the highest, indicating that it had a more generalized diet and richer food sources. 4) With Mytilus coruscus as a baseline organism, the trophic level of C. myriaster was the highest (3.38), followed by S. marmoratus (3.09), and the trophic level of O. fasciatus was the smallest (3.00). 5) Results of the stable isotope mixture model (SIAR) showed that POM was the main carbon source of the three species, contributing 57.4%, 57.9%, and 92.0% of the total, respectively. In addition, the contribution rate of SOM was also high for O. fasciatus and S. marmoratus, which was 21.5% and 33.9%, respectively. This study could provide basic information and reference for understanding trophic structure and marine food web in Zhongjiashan Islands.

GPR35 antagonist CID-2745687 attenuates anchorage-independent cell growth by inhibiting YAP/TAZ activity in colorectal cancer cells.

Background and purpose: GPR35, a member of the orphan G-protein-coupled receptor, was recently implicated in colorectal cancer (CRC). However, whether targeting GPR35 by antagonists can inhibit its pro-cancer role has yet to be answered. Experimental approach: We applied antagonist CID-2745687 (CID) in established GPR35 overexpressing and knock-down CRC cell lines to understand its anti-cell proliferation property and the underlying mechanism. Key results: Although GPR35 did not promote cell proliferation in 2D conditions, it promoted anchorage-independent growth in soft-agar, which was reduced by GPR35 knock-down and CID treatment. Furthermore, YAP/TAZ target genes were expressed relatively higher in GPR35 overexpressed cells and lower in GPR35 knock-down cells. YAP/TAZ activity is required for anchorage-independent growth of CRC cells. By detecting YAP/TAZ target genes, performing TEAD4 luciferase reporter assay, and examining YAP phosphorylation and TAZ protein expression level, we found YAP/TAZ activity is positively correlated to GPR35 expression level, which CID disrupted in GPR35 overexpressed cells, but not in GPR35 knock-down cells. Intriguingly, GPR35 agonists did not promote YAP/TAZ activity but ameliorated CID's inhibitory effect; GPR35-promoted YAP/TAZ activity was only partly attenuated by ROCK1/2 inhibitor. Conclusion and implications: GPR35 promoted YAP/TAZ activity partly through Rho-GTPase with its agonist-independent constitutive activity, and CID exhibited its inhibitory effect. GPR35 antagonists are promising anti-cancer agents that target hyperactivation and overexpression of YAP/TAZ in CRC.

Estimation and testing for clustered interval-censored bivariate survival data with application using the semi-parametric version of the Clayton-Oakes model.

The Kaplan-Meier estimator is ubiquitously used to estimate survival probabilities for time-to-event data. It is nonparametric, and thus does not require specification of a survival distribution, but it does assume that the risk set at any time t consists of independent observations. This assumption does not hold for data from paired organ systems such as occur in ophthalmology (eyes) or otolaryngology (ears), or for other types of clustered data. In this article, we estimate marginal survival probabilities in the setting of clustered data, and provide confidence limits for these estimates with intra-cluster correlation accounted for by an interval-censored version of the Clayton-Oakes model. We develop a goodness-of-fit test for general bivariate interval-censored data and apply it to the proposed interval-censored version of the Clayton-Oakes model. We also propose a likelihood ratio test for the comparison of survival distributions between two groups in the setting of clustered data under the assumption of a constant between-group hazard ratio. This methodology can be used both for balanced and unbalanced cluster sizes, and also when the cluster size is informative. We compare our test to the ordinary log rank test and the Lin-Wei (LW) test based on the marginal Cox proportional Hazards model with robust standard errors obtained from the sandwich estimator. Simulation results indicate that the ordinary log rank test over-inflates type I error, while the proposed unconditional likelihood ratio test has appropriate type I error and higher power than the LW test. The method is demonstrated in real examples from the Sorbinil Retinopathy Trial, and the Age-Related Macular Degeneration Study. Raw data from these two trials are provided.

Semiparametric predictive inference for failure data using first-hitting-time threshold regression.

The progression of disease for an individual can be described mathematically as a stochastic process. The individual experiences a failure event when the disease path first reaches or crosses a critical disease level. This happening defines a failure event and a first hitting time or time-to-event, both of which are important in medical contexts. When the context involves explanatory variables then there is usually an interest in incorporating regression structures into the analysis and the methodology known as threshold regression comes into play. To date, most applications of threshold regression have been based on parametric families of stochastic processes. This paper presents a semiparametric form of threshold regression that requires the stochastic process to have only one key property, namely, stationary independent increments. As this property is frequently encountered in real applications, this model has potential for use in many fields. The mathematical underpinnings of this semiparametric approach for estimation and prediction are described. The basic data element required by the model is a pair of readings representing the observed change in time and the observed change in disease level, arising from either a failure event or survival of the individual to the end of the data record. An extension is presented for applications where the underlying disease process is unobservable but component covariate processes are available to construct a surrogate disease process. Threshold regression, used in combination with a data technique called Markov decomposition, allows the methods to handle longitudinal time-to-event data by uncoupling a longitudinal record into a sequence of single records. Computational aspects of the methods are straightforward. An array of simulation experiments that verify computational feasibility and statistical inference are reported in an online supplement. Case applications based on longitudinal observational data from The Osteoarthritis Initiative (OAI) study are presented to demonstrate the methodology and its practical use.

Coupled folding-upon-binding of human tumor suppressor MIG6 to lung cancer EGFR kinase domain and molecular trimming/stapling of MIG6-derived ß-hairpins to target the coupling event.

Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured ß-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed ß-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.

[Guidelines for prevention and treatment of colorectal adenoma with integrated Chinese and western medicine].

The Guidelines for prevention and treatment of colorectal adenoma with integrated Chinese and western medicine are put forward by Nanjing University of Chinese Medicine and approved by China Association of Chinese Medicine. According to the formulation processes and methods of relevant clinical practice guidelines, the experts in clinical medicine and methodology were organized to discuss the key problems to be addressed in the clinical prevention and treatment of colorectal adenoma(CRA) and provided answers following the evidence-based medicine method, so as to provide guidance for clinical decision-making. CRA is the major precancerous disease of colorectal cancer. Although the prevention and treatment with integrated Chinese and western medicine have been applied to the clinical practice of CRA, there is still a lack of high-quality guidelines. Four basic questions, 15 clinical questions, and 10 outcome indicators were determined by literature research and Delphi questionnaire. The relevant randomized controlled trial(RCT) was retrieved from CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase, Cochrane Library, Web of Science, and 2 clinical trial registries, and finally several RCTs meeting the inclusion criteria were included. The data extracted from the RCT was imported into RevMan 5.3 for evidence synthesis, and the evidence was evaluated based on the Grading of Recommendations, Assessment, Development, and Evaluations(GRADE). The final recommendations were formed by the nominal group method based on the evidence summary table. The guidelines involve the diagnosis, screening, treatment with integrated Chinese and western medicine, prevention, and follow-up of colorectal adenoma, providing options for the clinical prevention and treatment of CRA.