Synthesis of Amides via the Amination of Aldehydes with Hydroxylamines Promoted by TBAF·3H2O.

A metal-free, mild, and efficient method for the synthesis of amides has been developed from the amination of aldehydes with hydroxylamines promoted by TBAF·3H2O in the presence of KOH. Control experiments showed that the nitrone was the intermediate of this amination. By this method, a series of amides, biologically active compounds bebenil and a COX inhibitor were obtained in moderate to good yields.

Innovative Biomaterials for Bone Tumor Treatment and Regeneration: Tackling Postoperative Challenges and Charting the Path Forward.

Surgical resection of bone tumors is the primary approach employed in the treatment of bone cancer. Simultaneously, perioperative interventions, particularly postoperative adjuvant anticancer strategies, play a crucial role in achieving satisfactory therapeutic outcomes. However, the occurrence of postoperative bone tumor recurrence, metastasis, extensive bone defects, and infection are significant risks that can result in unfavorable prognoses or even treatment failure. In recent years, there has been significant progress in the development of biomaterials, leading to the emergence of new treatment options for bone tumor therapy and bone regeneration. This progress report aims to comprehensively analyze the strategic development of unique therapeutic biomaterials with inherent healing properties and bioactive capabilities for bone tissue regeneration. These composite biomaterials, classified into metallic, inorganic non-metallic, and organic types, are thoroughly investigated for their responses to external stimuli such as light or magnetic fields, internal interventions including chemotherapy or catalytic therapy, and combination therapy, as well as their role in bone regeneration. Additionally, an overview of self-healing materials for osteogenesis is provided and their potential applications in combating osteosarcoma and promoting bone formation are explored. Furthermore, the safety concerns of integrated materials and current limitations are addressed, while also discussing the challenges and future prospects.

Morphological Changes and Strong Cytotoxicity in Yarrowia lipolytica by Overexpressing Delta-12-Desaturase.

In this study, delta-12 desaturase was overexpressed in Yarrowia lipolytica using the single-copy integrative vector pINA1312 and multicopy integrative vector pINA1292, resulting in the engineered yeast strains 1312-12 and 1292-12, respectively. The content of intracellular linoleic acid (LA) in the 1292-12 strain was much higher than in the 1312-12 strain and the control group. One interesting finding was that the 1292-12 strain showed obvious changes in surface morphology. The 1292-12 colonies were much smaller and smoother, whereas their single cells became much larger compared to the control strain. In addition, the dry cell weight (DCW) of the 1292-12 strain was obviously increased from 8.5 to 12.7 g/L, but the viable cell number sharply decreased from 107 to 105/mL. These results indicated that increased LA content in Yarrowia lipolytica could induce morphological changes or even oxidative stress-dependent cell death. The reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated in the 1292-12 strain, while the antioxidant activities of intracellular catalase (CAT) and superoxide dismutase (SOD) were significantly decreased by 27.6 and 32.0%, respectively. Furthermore, it was also revealed that these issues could be ameliorated by the exogenous supplementation of vitamin C, fish and colza oil.

Gut-lung axis: role of the gut microbiota in non-small cell lung cancer immunotherapy.

Immunotherapy for non-small cell lung cancer (NSCLC) has advanced considerably over the past two decades. In particular, immune checkpoint inhibitors are widely used for treating NSCLC. However, the overall cure and survival rates of patients with NSCLC remain low. Therefore, continuous investigation into complementary treatments is necessary to expand the clinical advantages of immunotherapy to a larger cohort of patients with NSCLC. Recently, the distinctive role of the gut microbiota (GM) in the initiation, progression, and dissemination of cancer has attracted increasing attention. Emerging evidence indicates a close relationship between the gut and lungs, known as the gut-lung axis (GLA). In this review, we aim to provide a comprehensive summary of the current knowledge regarding the connection between the GM and the outcomes of immunotherapy in NSCLC, with particular focus on the recent understanding of GLA. Overall, promising GM-based therapeutic strategies have been observed to improve the effectiveness or reduce the toxicity of immunotherapy in patients with NSCLC, thus advancing the utilization of microbiota precision medicine.

Association between genetically predicted rheumatoid arthritis and alopecia areata: a two-sample Mendelian randomization study.

Background: Although numerous observational studies have indicated a potential association between autoimmune diseases, such as rheumatoid arthritis (RA) and alopecia areata (AA), the research reports lack a clear causal relationship. In this study, our objective is to utilize the Mendelian randomization (MR) design to examine the potential causal association between RA and AA. Methods: To investigate the causal relationship between RA and AA, we utilized large-scale gene aggregation data from genome-wide association studies (GWAS), including RA (n=58,284) and AA (n=361,822) based on previous observational studies. In our analysis, we mainly employed the inverse variance-weighted (IVW) method of the random effects model, supplemented by the weighted median (WM) method and the MR Egger method. Results: The findings from the IVW methods revealed a significant association between genetically predicted RA and an increased likelihood of AA, as evidenced by an odds ratio of 1.21 (95%CI = 1.11-1.32; P < 0.001. Both the WM method and MR-Egger regression consistently showed significant directional outcomes (Both P < 0.05), indicating a robust association between RA and AA. Additionally, both the funnel plot and the MR-Egger intercepts provided evidence of the absence of directional pleiotropy, suggesting that the observed association is not influenced by other common genetic factors. Conclusions: The results of the study suggest a possible link between genetically predicted RA and AA. This finding highlights the importance for individuals diagnosed with RA to remain vigilant and aware of the potential development of AA. Regular monitoring and early detection can be crucial in managing and addressing this potential complication.

Unconventional Source of Neurotoxic Protein Aggregation from Organelle Off-Target Bax∆2 in Alzheimer's Disease.

Protein aggregates are a hallmark of Alzheimer's disease (AD). Extensive studies have focused on ß-amyloid plaques and Tau tangles. Here, we illustrate a novel source of protein aggregates in AD neurons from organelle off-target proteins. Bax is a mitochondrial pore-forming pro-death protein. What happens to Bax if it fails to target mitochondria? We previously showed that a mitochondrial target-deficient alternatively spliced variant, Bax∆2, formed large cytosolic protein aggregates and triggered caspase 8-mediated cell death. Bax∆2 protein levels were low in most normal organs and the proteins were quickly degraded in cancer. Here, we found that 85% of AD patients had Bax∆2 required alternative splicing. Increased Bax∆2 proteins were mostly accumulated in neurons of AD-susceptible brain regions. Intracellularly, Bax∆2 aggregates distributed independently of Tau tangles. Interestingly, Bax∆2 aggregates triggered the formation of stress granules (SGs), a large protein-RNA complex involved in AD pathogenesis. Although the functional domains required for aggregation and cell death are the same as in cancer cells, Bax∆2 relied on SGs, not caspase 8, for neuronal cell death. These results imply that the aggregation of organelle off-target proteins, such as Bax∆2, broadens the scope of traditional AD pathogenic proteins that contribute to the neuronal stress responses and AD pathogenesis.

Backdoor attacks against distributed swarm learning.

Traditional machine learning approaches often need a central server, where raw datasets or model updates are trained or aggregated in a centralized way. However, these approaches are vulnerable to many attacks, especially by the malicious server. Recently, a new distributed machine learning paradigm, called Swarm Learning (SL), has been proposed to support no-central-server based decentralized training. In each training round, each participant node has a chance to be selected to serve as a temporary server. Thus, these participant nodes do not need to share their private datasets to ensure a fair and secure model aggregation in a central server. To the best of our knowledge, there are no existing solutions about the security threats in swarm learning. In this paper, we investigate how to implant backdoor attacks against swarm learning to illustrate its potential security risk. Experiment results confirm the effectiveness of our method with high attack accuracies in different scenarios. We also study several defense methods to alleviate these backdoor attacks.

Expression profile of the proapoptotic protein Bax in the human brain.

Bax is a well-known universal proapoptotic protein. Bax protein is detected in almost all human organs, and its expression levels can be correlated with disease progression and therapeutic efficacy in certain settings. Interestingly, increasing evidence has shown that mature neuronal cell death is often not typical apoptosis. Most results on the expression of Bax proteins (predominantly Baxα) in the human brain come from disease-oriented studies, and the data on Bax protein expression in the normal brain are limited and lack consistency due to many variable factors. Here, we analyzed Bax RNA and protein expression data from multiple databases and performed immunostaining of over 80 samples from 25 healthy subjects across 7 different brain regions. We found that Bax protein expression was heterogeneous across brain regions and individual subjects. Both neurons and glial cells, such as astrocytes, could be Bax positive, but Bax positivity appeared to be highly selective, even within the same cell type in the same region. Furthermore, Bax proteins could be localized in the cytosol (evenly spread or concentrated to one region), nucleus or nucleolus depending on the cell type. Such variation and distribution in Bax expression suggest that Bax may function differently in the human brain than in other organs.

Homologous Overexpression of Diacylglycerol Acyltransferase in Oleaginous Fungus Mucor circinelloides WJ11 Enhances Lipid Accumulation under Static Solid Cultivation.

Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form triacylglycerol (TAG). Previous studies strongly indicate that DGAT2, rather than DGAT1, is crucial for TAG accumulation in the oleaginous fungus Mucor circinelloides. To increase the lipid content of M. circinelloides WJ11, McDGAT2 was overexpressed by homologous recombination; compared to the control strain Mc2075, transformants McDGAT2d showed a significant increase in biomass for both spores and mycelia (from 87.7 to 101.2 mg/g in spores and from 75.6 to 93.1 mg/g in mycelia). McDGAT2 overexpression under static solid fermentation gave a greater boost to lipid accumulation in mycelia than in spores. Total fatty acid content in mycelia increased by 68.0% (from 13.6 to 22.8%) and in spores by 26.3% (from 10.6 to 13.4%). However, under submerged fermentation, the lipid content of McDGAT2d was the same as the control, while biomass was slightly reduced. Transcriptomics showed that NADPH was derived mainly from the pentose phosphate pathway, acetyl-CoA was from multiple pathways, and leucine metabolism played an important role in substrate supply for fatty acid biosynthesis. Static solid fermentation may be the more suitable fermentation method for microbial oil production by filamentous fungi due to its lower fermentation costs.

In Vitro Phytobiological Investigation of Bioactive Secondary Metabolites from the Malus domestica-Derived Endophytic Fungus Aspergillus tubingensis Strain AN103.

Endophytic fungi including black aspergilli have the potential to synthesize multiple bioactive secondary metabolites. Therefore, the search for active metabolites from endophytic fungi against pathogenic microbes has become a necessity for alternative and promising strategies. In this study, 25 endophytic fungal isolates associated with Malus domestica were isolated, grown, and fermented on a solid rice medium. Subsequently, their ethyl acetate crude extracts were pretested for biological activity. One endophytic fungal isolate demonstrated the highest activity and was chosen for further investigation. Based on its phenotypic, ITS ribosomal gene sequences, and phylogenetic characterization, this isolate was identified as Aspergillus tubingensis strain AN103 with the accession number (KR184138). Chemical investigations of its fermented cultures yielded four compounds: Pyranonigrin A (1), Fonsecin (2), TMC 256 A1 (3), and Asperazine (4). Furthermore, 1H-NMR, HPLC, and LC-MS were performed for the identification and structure elucidation of these metabolites. The isolated pure compounds showed moderate-to-potent antibacterial activities against Pseudomonas aeruginosa and Escherichia coli (MIC value ranged from 31 and 121 to 14.5 and 58.3 µg/mL), respectively; in addition, the time−kill kinetics for the highly sensitive bacteria against isolated compounds was also investigated. The antifungal activity results show that (3) and (4) had the maximum effect against Fusarium solani and A. niger with inhibition zones of 16.40 ± 0.55 and 16.20 ± 0.20 mm, respectively, and (2) had the best effect against Candida albicans, with an inhibition zone of 17.8 ± 1.35 mm. Moreover, in a cytotoxicity assay against mouse lymphoma cell line L5178Y, (4) exhibited moderate cytotoxicity (49% inhibition), whereas (1−3) reported weak cytotoxicity (15, 26, and 19% inhibition), respectively. Our results reveal that these compounds might be useful to develop potential cytotoxic and antimicrobial drugs and an alternative source for various medical and pharmaceutical fields.

Mucor circinelloides: a model organism for oleaginous fungi and its potential applications in bioactive lipid production.

Microbial oils have gained massive attention because of their significant role in industrial applications. Currently plants and animals are the chief sources of medically and nutritionally important fatty acids. However, the ever-increasing global demand for polyunsaturated fatty acids (PUFAs) cannot be met by the existing sources. Therefore microbes, especially fungi, represent an important alternative source of microbial oils being investigated. Mucor circinelloides-an oleaginous filamentous fungus, came to the forefront because of its high efficiency in synthesizing and accumulating lipids, like γ-linolenic acid (GLA) in high quantity. Recently, mycelium of M. circinelloides has acquired substantial attraction towards it as it has been suggested as a convenient raw material source for the generation of biodiesel via lipid transformation. Although M. circinelloides accumulates lipids naturally, metabolic engineering is found to be important for substantial increase in their yields. Both modifications of existing pathways and re-formation of biosynthetic pathways in M. circinelloides have shown the potential to improve lipid levels. In this review, recent advances in various important metabolic aspects of M. circinelloides have been discussed. Furthermore, the potential applications of M. circinelloides in the fields of antioxidants, nutraceuticals, bioremediation, ethanol production, and carotenoids like beta carotene and astaxanthin having significant nutritional value are also deliberated.

Overexpression of Shinorhizobium meliloti flavohemoglobin improves cell growth and fatty acid biosynthesis in oleaginous fungus Mucor circinelloides.

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which deliver oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. The VHb and SHb genes were integrated into the M. circinelloides MU402 genome by homologous recombination. VHb and SHb protein expression was verified by carbon monoxide difference spectrum analysis. The biomass was increased by ~ 50% in the strain expressing SHb compared with VHb. The total fatty acid (TFA) content of the strain expressing SHb reached 15.7% of the dry cell weight (~ 40% higher than that of the control strain) during flask cultivation. The biomass and TFA content were markedly increased (12.1 g/L and 21.1% dry cell weight, respectively) in strains expressing SHb than strains expressing VHb during fermenter cultivation. VHb and SHb expression also increased the proportion of polyunsaturated fatty acids. Overexpressed bacterial hemoglobins, especially SHb, increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb improves fatty acid production more effectively than VHb in oleaginous microorganisms.

Oxidation of BINOLs by Hypervalent Iodine Reagents: Facile Synthesis of Xanthenes and Lactones.

Xanthene derivatives have broad applications in medicines, fluorescent probes, dyes, food additives, etc. Therefore, much attention was focused on developing the synthetic methods to prepare these compounds. Binaphthyl-based xanthene derivatives were prepared through the oxidation of BINOLs promoted by the hypervalent iodine reagent iodosylbenzene (PhIO). Nine-membered lactones were obtained through a similar oxidative reaction when iodoxybenzene (PhIO2 ) was used. Additionally, one-pot reactions of BINOLs, PhIO and nucleophiles such as alcohols and amines were also investigated to provide alkoxylated products and amides in good to excellent yields.

Chiral Ligands in Hypervalent Iodine Compounds: Synthesis and Structures of Binaphthyl-Based λ3 -Iodanes.

Several novel binaphthyl-based chiral hypervalent iodine(III) reagents have been prepared and structurally analysed. Various asymmetric oxidative reactions were applied to evaluate the reactivities and stereoselectivities of those reagents. Moderate to excellent yields were observed; however, very low stereoselectivities were obtained. NMR experiments indicated that these reagents are very easily hydrolysed in either chloroform or DMSO solvents leading to the limited stereoselectivities. It is concluded that the use of chiral ligands is an unsuccessful way to prepare efficient stereoselective iodine(III) reagents.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production.

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Ubl4A is critical for mitochondrial fusion process under nutrient deprivation stress.

Mitochondrial fusion and fission are dynamic processes regulated by the cellular microenvironment. Under nutrient starvation conditions, mitochondrial fusion is strengthened for energy conservation. We have previously shown that newborns of Ubl4A-deficient mice were more sensitive to starvation stress with a higher rate of mortality than their wild-type littermates. Ubl4A binds with the actin-related protein Arp2/3 complex to synergize the actin branching process. Here, we showed that deficiency in Ubl4A resulted in mitochondrial fragmentation and apoptosis. A defect in the fusion process was the main cause of the mitochondrial fragmentation and resulted from a shortage of primed Arp2/3 complex pool around the mitochondria in the Ubl4A-deficient cells compared to the wild-type cells. As a result, the mitochondrial fusion process was not undertaken quickly enough to sustain starvation stress-induced cell death. Consequently, fragmented mitochondria lost their membrane integrity and ROS was accumulated to trigger caspase 9-dependent apoptosis before autophagic rescue. Furthermore, the wild-type Ubl4A, but not the Arp2/3-binding deficient mutant, could rescue the starvation-induced mitochondrial fragmentation phenotype. These results suggest that Ubl4A promotes the mitochondrial fusion process via Arp2/3 complex during the initial response to nutrient deprivation for cell survival.

Harnessing C/N balance of Chromochloris zofingiensis to overcome the potential conflict in microalgal production.

Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.

Immunohistochemical detection of the pro-apoptotic Bax∆2 protein in human tissues.

The pro-apoptotic Bax isoform Bax∆2 was originally discovered in cancer patients with a microsatellite guanine deletion (G8 to G7). This deletion leads to an early stop codon; however, when combined with the alternative splicing of exon 2, the reading frame is restored allowing production of a full-length protein (Bax∆2). Unlike the parental Baxα, Bax∆2 triggers apoptosis through a non-mitochondrial pathway and the expression in human tissues was unknown. Here, we analyzed over 1000 tissue microarray samples from 13 different organs using immunohistochemistry. Bax∆2-positive cells were detected in all examined organs at low rates (1-5%) and mainly scattered throughout the connective tissues. Surprisingly, over 70% of normal colon samples scored high for BaxΔ2-positive staining. Only 7% of malignant colon samples scored high, with most high-grade tumors being negative. A similar pattern was observed in most organs examined. We also showed that both Baxα and Bax∆2 can co-exist in the same cells. Genotyping showed that the majority of Bax∆2-positive normal tissues contain no G7 mutation, but an unexpected high rate of G9 was observed. Although the underlying mechanism remains to be explored, the inverse correlation of Bax∆2 expression with tissue malignancy suggests that it may have a clinical implication in cancer development and treatment.

Mitochondrial Citrate Transport System in the Fungus Mucor circinelloides: Identification, Phylogenetic Analysis, and Expression Profiling During Growth and Lipid Accumulation.

The mitochondrial citrate transport system, composed of citrate and malate transporters (MTs), can regulate the citrate efflux from mitochondria to cytosol, and then citrate is cleaved into OAA and acetyl-CoA which can be used for fatty acid (FA) biosynthesis. However, in the fungus Mucor circinelloides the molecular mechanism of citrate efflux from the mitochondria by this system and its role in FA synthesis is unclear. In the present study, we have analyzed the genome of high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 to find the potential genes involving in this system. Five potential genes are present in the genome of WJ11. These genes encode one citrate transport protein (CT), one tricarboxylate carrier (TCT), one MT, and two 2-oxoglutarate:malate antiporters (SoDIT-a and SoDIT-b). However, the genome of CBS 277.49 contains the same set of genes, except for the presence of just one SoDIT. The proteins from WJ11 had similar properties as their counterparts in CBS 277.49. Moreover, phylogenetic analyses revealed the evolutionary relationship of these proteins and illuminated their typical motifs related to potential functions. Additionally, the expression of these genes was analyzed to predict the possible functions in lipid metabolism in M. circinelloides. This is the first study to report the in silico analysis of structures and functions of the mitochondrial citrate transport system in M. circinelloides. This work showed a new strategy for research for the selection of candidate genes for further detailed functional investigation of the mitochondrial citrate transport system in lipid accumulation.

Discovery and characterization of a stable lipase with preference toward long-chain fatty acids.

OBJECTIVES: To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database. RESULTS: Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 °C, and stability between 40 and 50 °C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates. CONCLUSION: The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.