ASIC1a regulates ferroptosis in hepatic stellate cells via the Hippo/Yap-1 pathway in liver fibrosis.

BACKGROUND: Liver fibrosis is a sustained process of liver tissue damage and repair caused by various physiological and pathological factors, with the activation and proliferation of hepatic stellate cells being central. Therefore, understanding and clarifying the relevant mechanisms of hepatic stellate cell activation and death is of great clinical significance for the treatment of liver fibrosis diseases. METHODS: In vivo, recombinant adeno-associated virus was used to infect the liver of experimental mice, overexpressing ASIC1a, and based on this, a liver fibrosis model treated with sorafenib was constructed. In vitro, using RNA plasmid technology to transfect HSC-T6 cells, ASIC1a was overexpressed or silenced in the cells, and on this basis, PDGF-BB and Sorafenib were used to stimulate HSC-T6 cells, causing activated HSC-T6 to undergo ferroptosis. RESULTS: The ferroptosis inducers Sorafenib and erastin can induce ferroptosis in HSCs, effectively inhibiting or reversing the progression of liver fibrosis. We found that the expression level of ASIC1a was significantly reduced in the livers of mice with liver fibrosis treated with Sorafenib. After treatment with an adeno-associated virus overexpressing ASIC1a, the therapeutic effect of Sorafenib was inhibited, and the level of ferroptosis induced by Sorafenib was also inhibited. The induction of ferroptosis in hepatic stellate cells in vitro depends on the presence of ASIC1a. By further exploring the potential mechanism, we observed that the overexpression of ASIC1a can promote an increase in YAP nuclear translocation, thereby regulating the activity of Hippo/YAP pathway signaling. After treatment with Sorafenib, the influx of Ca2+ significantly increased when ASIC1a was overexpressed, and BAPTA-AM intervention eliminated the intracellular Ca2+ accumulation induced by ASIC1a overexpression. CONCLUSIONS: This indicated that the activation of YAP depends on the calcium ion influx induced by ASIC1a, which regulates ferroptosis in hepatic stellate cells by regulating the calcium ion-dependent Hippo/YAP pathway.

Biliverdin improved angiogenesis and suppressed apoptosis via PI3K/Akt-mediated Nrf2 antioxidant system to promote ischemic flap survival.

Plastic and reconstructive surgeons frequently utilize random skin flap transplantation to repair skin defects. However, the procedure carries a substantial risk of necrosis. Previous research has suggested that Biliverdin (Bv), the main component of Calculus Bovis, possessed potent anti-ischemic properties, making it a potential therapeutic agent for skin flap survival. Hence, in this study, the potential of Bv in promoting flap survival has been comprehensively investigated. Network pharmacology analysis revealed that the pharmacological effects of Bv on ischemic diseases may be attributed to its modulation of various signaling molecules, including the PI3K-Akt pathway. In vitro results demonstrated that Bv treatment significantly promoted angiogenesis in human umbilical vein endothelial cells (HUVEC), even in the presence of H2O2. This was evident by the increased cell proliferation, enhanced migration, and improved tube formation. Bv also effectively attenuated the intracellular generation of reactive oxygen species (ROS) induced by H2O2, which was achieved by suppressing mitochondrial ROS production through the PI3K/Akt-mediated activation of Nrf2/HO-1 signaling pathway. Consequently, Bv treatment led to a significant reduction in apoptosis and an increase in cell viability of HUVEC. Furthermore, in vivo experiment demonstrated that Bv treatment vastly elevated flap survival through enhancing angiogenesis while decreasing oxidative stress and apoptosis, which was comparable to the results of positive control of N-acetylcysteine (Nac). In conclusion, this study not only established a solid foundation for future study on therapeutic potential of Bv, but also proposed a promising treatment approach for enhancing the success rate of flap transplants and other ischemic-related tissue repair.

CCC pincer Ru complex-catalyzed C-H vinylation/6π-E-cyclization of aldimines for constructing 4H-pyrido[1,2-a]pyrimidines.

An unusual cascade C-H activation, vinylation and 6π-electrocyclization of 2-pyridyl aldimines with vinyl bromides/triflates was achieved using catalysis with a unique CCC pincer NHC-Ru(iii) complex (Cat B). This reaction was found to enable a rapid and diverse synthesis of polycyclic 4H-pyrido[1,2-a]pyrimidine derivatives in mostly good to high yields, and with a broad substrate scope. A mechanistic study suggested the formation of a semi-opened Ru(iii) intermediate chelating/activating the aldimine, and the occurrence of single-electron transfer (SET) to generate a vinyl radical, followed by vinylation and then an intramolecular 6π-electrocyclization of 1N,3N-hexatrene to form the product. This protocol provides a convenient approach for preparing and seeking new drug candidates.

An integrative pan-cancer analysis of MASP1 and the potential clinical implications for the tumor immune microenvironment.

Mannose-binding lectin-associated serine protease 1 (MASP1) plays a crucial role in the complement lectin pathway and the mediation of immune responses. However, comprehensive research on MASP1 across various cancer types has not been performed to date. This study aimed to evaluate the significance of MASP1 in pan-cancer. The Cancer Genome Atlas (TCGA), UCSC Xena and Genotype Tissue Expression (GTEx) databases were used to evaluate the expression profiles, genomic features, prognostic relevance, and immune microenvironment associations of MASP1 across 33 cancer types. We observed significant dysregulation of MASP1 expression in multiple cancers, with strong associations between MASP1 expression levels and diagnostic value as well as patient prognosis. Mechanistic insights revealed significant correlations between MASP1 levels and various immunological and genomic factors, including tumor-infiltrating immune cells (TIICs), immune-related genes, mismatch repair (MMR), tumor mutation burden (TMB), and microsatellite instability (MSI), highlighting a critical regulatory function of MASP1 within the tumor immune microenvironment (TIME). In vitro and in vivo experiments demonstrated that MASP1 expression was markedly decreased in liver hepatocellular carcinoma (LIHC). Moreover, the overexpression of MASP1 in hepatocellular carcinoma (HCC) cell lines significantly inhibited their proliferation, invasion and migration. In conclusion, MASP1 exhibits differential expression in the pan-cancer analyses and might play an important role in TIME. MASP1 is a promising prognostic biomarker and a potential target for immunological research, particularly in LIHC.

Design and Optimization of Iron-Based Superionic-Like Conductor Anode for High-Performance Lithium/Sodium-Ion Batteries.

Metal selenides have received extensive research attention as anode materials for batteries due to their high theoretical capacity. However, their significant volume expansion and slow ion migration rate result in poor cycling stability and suboptimal rate performance. To address these issues, the present work utilized multivalent iron ions to construct fast pathways similar to superionic conductors (Fe-SSC) and introduced corresponding selenium vacancies to enhance its performance. Based on first-principles calculations and molecular dynamics simulations, it is demonstrated that the addition of iron ions and the presence of selenium vacancies reduced the material's work function and adsorption energy, lowered migration barriers, and enhances the migration rate of Li+ and Na+. In Li-ion half batteries, this composite material exhibites reversible capacity of 1048.3 mAh g-1 at 0.1 A g-1 after 100 cycles and 483.6 mAh g-1 at 5.0 A g-1 after 1000 cycles. In Na-ion half batteries, it is 687.7 mAh g-1 at 0.1 A g-1 after 200 cycles and 325.9 mAh g-1 at 5.0 A g-1 after 1000 cycles. It is proven that materials based on Fe-SSC and selenium vacancies have great applications in both Li-ion batteries and Na-ion batteries.

Single-cell multi-modal chromatin profiles revealing epigenetic regulations of cells in hepatocellular carcinoma.

BACKGROUND: Various epigenetic regulations systematically govern gene expression in cells involving various biological processes. Dysregulation of the epigenome leads to aberrant transcriptional programs and subsequently results in diseases, such as cancer. Therefore, comprehensive profiling epigenomics is essential for exploring the mechanisms underlying gene expression regulation during development and disease. METHODS: In this study, we developed single-cell chromatin proteins and accessibility tagmentation (scCPA-Tag), a multi-modal single-cell epigenetic profile capturing technique based on barcoded Tn5 transposases and a droplet microfluidics platform. scCPA-Tag enables the simultaneous capture of DNA profiles of histone modification and chromatin accessibility in the same cell. RESULTS: By applying scCPA-Tag to K562 cells and a hepatocellular carcinoma (HCC) sample, we found that the silence of several chromatin-accessible genes can be attributed to lysine-27-trimethylation of the histone H3 tail (H3K27me3) modification. We characterized the epigenetic features of the tumour cells and different immune cell types in the HCC tumour tissue by scCPA-Tag. Besides, a tumour cell subtype (C2) with more aggressive features was identified and characterized by high chromatin accessibility and a lower abundance of H3K27me3 on tumour-promoting genes. CONCLUSIONS: Our multi-modal scCPA-Tag provides a comprehensive approach for exploring the epigenetic landscapes of heterogeneous cell types and revealing the mechanisms of gene expression regulation during developmental and pathological processes at the single-cell level. HIGHLIGHTS: scCPA-Tag offers a highly efficient and high throughput technique to simultaneously profile histone modification and chromatin accessibility within a single cell. scCPA-Tag enables to uncover multiple epigenetic modification features of cellular compositions within tumor tissues. scCPA-Tag facilitates the exploration of the epigenetic landscapes of heterogeneous cell types and provides the mechanisms governing gene expression regulation.

A novel experimental mouse model of diabetic nonalcoholic steatohepatitis: A critical role for acid-sensitive Ion Channel 1a.

BACKGROUND: A two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model. METHODS: The combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model. RESULTS: The mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy. CONCLUSION: A FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH.

Identification and validation of ferroptosis-related hub genes and immune infiltration in liver ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a cumulation of pathophysiological processes that involves cell and organelle damage upon blood flow constraint and subsequent restoration. However, studies on overall immune infiltration and ferroptosis in liver ischemia-reperfusion injury (LIRI) are limited. This study explored immune cell infiltration and ferroptosis in LIRI using bioinformatics and experimental validation. The GSE151648 dataset, including 40 matched pairs of pre- and post- transplant liver samples was downloaded for bioinformatic analysis. Eleven hub genes were identified by overlapping differentially expressed genes (DEGs), iron genes, and genes identified through weighted gene co-expression network analysis (WGCNA). Subsequently, the pathway enrichment, transcription factor-target, microRNA-mRNA and protein-protein interaction networks were investigated. The diagnostic model was established by logistic regression, which was validated in the GSE23649 and GSE100155 datasets and verified using cytological experiments. Moreover, several drugs targeting these genes were found in DrugBank, providing a more effective treatment for LIRI. In addition, the expression of 11 hub genes was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in liver transplantation samples and animal models. The expression of the 11 hub genes increased in LIRI compared with the control. Five genes were significantly enriched in six biological process terms, six genes showed high enrichment for LIRI-related signaling pathways. There were 56 relevant transcriptional factors and two central modules in the protein-protein interaction network. Further immune infiltration analysis indicated that immune cells including neutrophils and natural killer cells were differentially accumulated in the pre- and post-transplant groups, and this was accompanied by changes in immune-related factors. Finally, 10 targeted drugs were screened. Through bioinformatics and further experimental verification, we identified hub genes related to ferroptosis that could be used as potential targets to alleviate LIRI.

Essential roles of histone lysine methyltransferases EZH2 and EHMT1 in male embryo development of Phenacoccus solenopsis.

Paternal genome elimination (PGE) is an intriguing but poorly understood reproductive strategy in which females are typically diploid, but males lose paternal genomes. Paternal genome heterochromatin (PGH) occurs in arthropods with germline PGE, such as the mealybug, coffee borer beetles, and booklice. Here, we present evidence that PGH initially occurs during early embryo development at around 15 h post-mating (hpm) in the cotton mealybug, Phenacoccus solenopsis Tinsley. Transcriptome analysis followed by qPCR validation indicated that six histone lysine methyltransferase (KMT) genes are predominantly expressed in adult females. We knocked down these five genes through dsRNA microinjection. We found that downregulation of two KMT genes, PsEZH2-X1 and PsEHMT1, resulted in a decrease of heterochromatin-related methylations, including H3K27me1, H3K27me3, and H3K9me3 in the ovaries, fewer PGH male embryos, and reduced male offspring. For further confirmation, we obtained two strains of transgenic tobacco highly expressing dsRNA targeting PsEZH2-X1 and PsEHMT1, respectively. Similarly, fewer PGH embryos and fewer male offspring were observed when feeding on these transgenic tobacco plants. Overall, we present evidence that PsEZH2-X1 and PsEHMT1 have essential roles in male embryo survival by regulating PGH formation in cotton mealybugs.

TONSL promotes lung adenocarcinoma progression, immune escape and drug sensitivity.

PURPOSE: The tonsoku-like DNA repair protein (TONSL) encoded by the TONSL gene, located on chromosome 8q24.3, is crucial for repairing DNA double-strand breaks through homologous recombination. However, TONSL overexpression in lung adenocarcinoma (LUAD) promotes tumor development, leading to a poor prognosis. METHODS: TONSL was verified as a reliable prognostic marker for LUAD using bioinformatics, and clinical features related to LUAD prognosis were screened from the TCGA database to establish the relationship between risk factors and TONSL expression. In addition, TONSL expression in normal and LUAD tissues was verified using real-time quantitative polymerase chain reaction and immunohistochemistry. To elucidate the possible functions of TONSL, TONSL-related differentially expressed genes were screened, and functional enrichment analysis was performed. Subsequently, siRNA was used to knock down TONSL expression in lung cancer cells for cytobehavioral experiments. The effects of TONSL expression on tumor immune escape were analyzed using the ESTIMATE algorithm and tumor immune-infiltration analysis. In addition, the half-maximal inhibitory concentration of LUAD with varying TONSL expression levels in response to first-line chemotherapeutic drugs and epidermal growth factor receptor-tyrosine kinase inhibitors was analyzed for drug sensitivity. RESULTS: Up-regulation of TONSL in LUAD promotes the proliferation, migration, and invasion of lung cancer cells, thereby contributing to a poor prognosis. Furthermore, TONSL overexpression promotes immune escape and drug sensitivity in LUAD. CONCLUSION: TONSL serves as a reliable prognostic marker for LUAD, and its up-regulation is associated with increased immune escape and drug sensitivity. These findings suggest that TONSL holds potential as a novel therapeutic target for LUAD.

Shallow tillage mitigates plant competition by increasing diversity and altering plant community assembly process.

Introduction: Understanding how human activities affect biodiversity is needed to inform systemic policies and targets for achieving sustainable development goals. Shallow tillage to remove Artemisia ordosica is commonly conducted in the Mu Us Desert. However, the impacts of shallow tillage on plant community species diversity, phylogenetic structure, and community assembly processes remain poorly understood. Methods: This study explores the effects of shallow tillage on species diversity including three a-diversity and two b-diversity indicators, as well as phylogenetic structure [phylogenetic diversity (PD), net relatedness index (NRI), and nearest taxon index (NTI)]. Additionally, this research analyzes the effects of shallow tillage on the community assembly process. Results and discussion: The results showed that the a-diversity index, b-diversity index, and PD of the shallow tillage (ST) communities were significantly higher than those of the non-shallow tillage (NT) communities, and the phylogenetic structures of both the ST and NT communities tended to be differentiated, with competitive exclusion being the main mechanism of plant assembly. However, shallow tillage increased the relative importance of the stochastic processes dominated by dispersal limitation, mitigating plant competition in the communities. This conclusion was supported by the Raup-Crick difference index-based analysis. Conclusion: Therefore, for the ecological restoration of the Mu Us Desert, species with adaptability and low niche overlap should be selected to increase the utilization efficiency of the environmental resources. The results of this study provide a foundation for policy development for ecosystem management and restoration in the Mu Us Desert.

Comparative time-series multi-omics analyses suggest H1.2 involvement in anoxic adaptation and cancer resistance.

The naked mole rat (NMR), Heterocephalus glaber, is known as the longest-lived rodent and is extraordinarily resistant to hypoxia and cancer. Here, both NMR embryonic fibroblasts (NEFs) and their mouse counterparts (MEFs) were subjected to anoxic conditions (0% O2, 5% CO2). A combination of comparative transcriptomics and proteomics was then employed to identify differentially expressed genes (DEGs). Notably, we observed distinct levels of histone H1.2 (encoded by HIST1H1C) accumulation between NEFs and MEFs. Subsequent mechanistic analyses showed that higher H1.2 expression in NEFs was associated with the lower expression of its inhibitor, PARP1. Additionally, we discovered that H1.2 can directly interact with HIF-1α PAS domains, thereby promoting the expression of HIF-1α through facilitating the dimerization with HIF-1ß. The overexpression of H1.2 was also found to trigger autophagy and to suppress the migration of cancer cells, as well as the formation of xenograft tumors, via the NRF2/P62 signaling pathway. Moreover, an engineered H1.2 knock-in mouse model exhibited significantly extended survival in hypoxic conditions (4% O2) and showed a reduced rate of tumor formation. Collectively, our results indicate a potential mechanistic link between H1.2 and the dual phenomena of anoxic adaptation and cancer resistance.

Transcriptome analysis of the medicinal mushroom Sanghuangporus vaninii in response to white light stress.

Light is a vital environmental factor that promotes the growth and development of edible fungi mycelium. Under white light, the mycelium color of Sanghuangporus vaninii shifts during its growth stages. To investigate the impact of visible light on mycelial morphogenesis, a comparative transcriptomic analysis was conducted. This analysis revealed the molecular processes that underpin mycelial growth and development in S. vaninii when cultured in both darkness and light conditions. From the analysis, 13,643 genes were aligned using Illumina raw reads. Of these, 596 genes exhibited significant expression changes under white light exposure. Specifically, 226 genes were upregulated and 370 downregulated, spanning 55 different metabolic pathways. We further classified differentially expressed genes (DEGs), these genes play roles in photomorphogenesis, signal transduction, carbohydrate metabolism, and melanin production, among other processes. Some are also implicated in cell cycle regulation and the differential expression of respiratory functions. The validation of the differentially expressed transcripts using qRT-PCR showed complete agreement with RNA-Seq data for 9 transcripts. Meanwhile, the light had an inhibitory effect on the bioactive components in S. vaninii. These findings offer valuable insights into the transcriptional shifts and molecular mechanisms driving the color change in S. vaninii under light exposure, providing a basis for further research into mechanisms of light-response regulation.

Contrasting effects of NADPH oxidases on the fungal hyphae growth and immune responses in Pleurotus ostreatus.

Pleurotus ostreatus is one of the most consumed mushroom species, as it serves as a high-quality food, favors a rich secondary metabolism, and has remarkable adaptability to the environment and predators. In this study, we investigated the function of two key reactive oxygen species producing enzyme NADPH oxidase (PoNoxA and PoNoxB) in P. ostreatus hyphae growth, metabolite production, signaling pathway activation, and immune responses to different stresses. Characterization of the Nox mutants showed that PoNoxB played an important role in the hyphal formation of the multicellular structure, while PoNoxA regulated apical dominance. The ability of P. ostreatus to tolerate a series of abiotic stress conditions (e.g., osmotic, oxidative, membrane, and cell-wall stresses) and mechanical damage repair was enhanced with PoNoxA over-expression. PoNoxB had a greater responsibility in regulating the polysaccharide composition of the cell wall and methyl jasmonate and gibberellin GA1 biosynthesis, and improved mushroom resistance against Tyrophagus putrescentiae. Moreover, mutants were involved in the jasmonate and GA signaling pathway, and toxic protein defense metabolite production. Our findings shed light on how the oyster mushroom senses stress signals and responds to adverse environments by the complex regulators of Noxs.

Asymmetric Intramolecular Amination Catalyzed with Cp*Ir-SPDO via Nitrene Transfer for Synthesis of Spiro-Quaternary Indolinone.

An asymmetric intramolecular spiro-amination to high steric hindering α-C-H bond of 1,3-dicarbonyl via nitrene transfer using inactive aryl azides has been carried out by developing a novel Cp*Ir(III)-SPDO (spiro-pyrrolidine oxazoline) catalyst, thereby enabling the first successful construction of structurally rigid spiro-quaternary indolinone cores with moderate to high yields and excellent enantioselectivities. DFT computations support the presence of double bridging H-F bonds between [SbF6]- and both the ligand and substrate, which favors the plane-differentiation of the enol π-bond for nitrenoid attacking. These findings open up numerous opportunities for the development of new asymmetric nitrene transfer systems.

The impacts of percutaneous coronary intervention to treat chronic total occlusion of right coronary artery on the 5-year prognosis: A single-centered retrospective study.

BACKGROUND: Chronic total occlusions (CTO) occur in about 20% of patients referred for coronary angiography, and right coronary artery (RCA) CTO has been reported in 38-50% of the entire CTO population. Limited data on angiographic and procedural characteristics of RCA-CTO and the risk of adverse cardiac events asks for a detailed study. METHODS: From 2010 to 2013, patients with attempted revascularization of at least one CTO lesion were included and followed up to 5 years after PCI. Eligible patients are assigned to RCA-CTO and non-RCA-CTO groups based on their target vessels. The primary endpoint was major adverse cardiovascular events (MACEs; a composite of all-cause death, myocardial infarction (MI) or rehospitalization for heart failure), and secondary endpoints were cardiac death, target lesion revascularization (TLR) and target vessel revascularization (TVR). RESULTS: The present study included 2659 eligible patients, among which 1285 patients were assigned to the RCA-CTO group, whereas 1374 patients were assigned to the non-RCA-CTO group. Lesions in RCA had longer lesion length, higher J-CTO score, higher rates of severe vessel tortuosity, a higher percentage of Rentrop grade 2-3, and more likely to be re-try lesion than those in LAD or LCX (all P < 0.01). CTO lesions in RCA reached less successful recanalization and post-procedural TIMI 3 flow (all <0.01). Multivariate Cox analysis revealed that RCA-CTO was not associated with primary outcome MACEs. Besides MACEs, RCA-CTO was also not associated with cardiac death, but was significantly associated with TLR and TVR (adjusted HR: 1.37 [95% CI:1.07-1.76], P = 0.01; adjusted HR: 1.43 [95% CI:1.13-1.82], P = 0.003). CONCLUSION: RCA-CTO lesions, which had more complex angiographic features, independently contributed to TLR and TVR but not to MACEs or cardiac death in the 5 years of follow-up.

Collapsed Star Copolymers Exhibiting Near Perfect Mimicry of the Therapeutic Protein "TRAIL".

Here we introduce amphiphilic star polymers as versatile protein mimics capable of approximating the activity of certain native proteins. Our study focuses on designing a synthetic polymer capable of replicating the biological activity of TRAIL, a promising anticancer protein that shows very poor circulation half-life. Successful protein mimicry requires precise control over the presentation of receptor-binding peptides from the periphery of the polymer scaffold while maintaining enough flexibility for protein-peptide binding. We show that this can be achieved by building hydrophobic blocks into the core of a star-shaped polymer, which drives unimolecular collapse in water. By screening a library of diblock copolymer stars, we were able to design structures with IC50's of ∼4 nM against a colon cancer cell line (COLO205), closely approximating the activity of the native TRAIL protein. This finding highlights the broad potential for simple synthetic polymers to mimic the biological activity of complex proteins.