Multimodal mechanisms of pathogenic variants in the signal peptide of FIX leading to hemophilia B.

Signal peptide (SP) is essential for protein secretion, and pathogenic variants in the SP of FIX have been identified in hemophilia B (HB). However, the underlying mechanism for the genotype-phenotype correlation of these variants has not been well studied. Here we systematically examined the effects of 13 pathogenic point variants in the SP of FIX using different approaches. Our results showed that these point variants lead to HB by missense variants and/or aberrant pre-mRNA splicing. The missense variants in h-region mainly affected the co-translational translocation function of the SP, and those in c-region caused FIX deficiency mainly by disturbing the co-translational translocation and/or cleavage of the SP. Almost absolute aberrant pre-mRNA splicing was only observed in variants of c.82T>G, but a slight change of splicing patterns was found in variants of c.53G>T, c.77C>A, c.82T>C, and c.83G>A, indicating that these variants might have different degree to affect pre-mRNA splicing. Although two 6-nt deletion aberrant pre-mRNA splicing products caused FIX deficiency by disturbing the SP cleavage, but they could produce some functional mature FIX and vitamin K could increase the secretion of functional FIX. Taken together, our data indicated that pathogenic variants in the SP of FIX caused HB through diverse molecular mechanisms or even a mixture of several mechanisms, and vitamin K availability could be partially attributed to varying bleeding tendencies in patients carrying the same variant in the SP.

Correction: Liu et al. The Discovery of Novel Agents against Staphylococcus aureus by Targeting Sortase A: A Combination of Virtual Screening and Experimental Validation. Pharmaceuticals 2024, 17, 58.

In the original publication [...].

Molecular basis of inherited protein C deficiency results from genetic variations in the signal peptide and propeptide regions.

BACKGROUND: Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC's signal peptide and propeptide have been reported in patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear. OBJECTIVES: To investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC's signal peptide and propeptide. METHODS: Using cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay. RESULTS: Our data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing. CONCLUSION: Our findings suggest that variations in PC's signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD.

The Discovery of Novel Agents against Staphylococcus aureus by Targeting Sortase A: A Combination of Virtual Screening and Experimental Validation.

Staphylococcus aureus (S. aureus), commonly known as "superbugs", is a highly pathogenic bacterium that poses a serious threat to human health. There is an urgent need to replace traditional antibiotics with novel drugs to combat S. aureus. Sortase A (SrtA) is a crucial transpeptidase involved in the adhesion process of S. aureus. The reduction in virulence and prevention of S. aureus infections have made it a significant target for antimicrobial drugs. In this study, we combined virtual screening with experimental validation to identify potential drug candidates from a drug library. Three hits, referred to as Naldemedine, Telmisartan, and Azilsartan, were identified based on docking binding energy and the ratio of occupied functional sites of SrtA. The stability analysis manifests that Naldemedine and Telmisartan have a higher binding affinity to the hydrophobic pockets. Specifically, Telmisartan forms stable hydrogen bonds with SrtA, resulting in the highest binding energy. Our experiments prove that the efficiency of adhesion and invasion by S. aureus can be decreased without significantly affecting bacterial growth. Our work identifies Telmisartan as the most promising candidate for inhibiting SrtA, which can help combat S. aureus infection.

Signet-Ring Cutaneous Metastasis Presenting with Huge Bunches of Grapes.

Signet-ring cell (SRC) is a histologic type in which cells show unique features under the microscope. We mainly found signet-ring cells (SRCs) in gastrointestinal and breast tumors. Cutaneous metastasis from internal carcinomas was an uncommon presentation. The cases of signet-ring cell carcinoma (SRCC) metastasis to the skin were rarely reported. Cutaneous metastasis indicated a poor prognosis for a patient. Here, we report a female who had huge grape-like nodules arising from gastrointestinal SRCC in her trunk and thigh.

Potential roles of gut microbial tryptophan metabolites in the complex pathogenesis of acne vulgaris.

Acne vulgaris is a chronic inflammatory skin disease in which the influence of gut microbiota has been implicated but without clarification of mechanisms. Gut microbiota may exert such an influence via metabolites, particularly those of tryptophan. End metabolites of tryptophan activate receptors, including aryl hydrocarbon, G protein-coupled, and pregnane X receptors to stabilize the immune microenvironment and intestinal mucosal homeostasis. Any impact on the pathogenesis of acne vulgaris remains unclear. The current review collates recent advances concerning potential roles of tryptophan metabolism in mediating skin inflammation, follicular sebaceous gland function and intestinal permeability, all of which influence the pathogenesis of acne vulgaris. The aim was to improve understanding of the pathogenesis of acne vulgaris and to expose therapeutic opportunities.

Utilization of gasification slag and petrochemical incineration fly ash for glass ceramic production.

This study investigated glass ceramics produced using coal gasification slag (CGS) and petrochemical incineration fly ash (PIFA) to immobilize hazardous heavy metals such as Cr and As. However, the crystallization kinetics and stabilization behavior mechanism of different heavy metals in the petrochemical incineration fly ash-derived glass-ceramics remains unclear. And X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and inductively coupled plasma mass spectrometry were used to characterize glass and crystalline products. In this paper, we reported the crystallization kinetics and chemical leaching characteristics of the glass ceramic. A low crystallization activation energy of 121.49 kJ/mol was achieved from crystallization peak of several different heating rates around 850°C, implying that it is easier to produce the glass ceramics at that temperature. The Avrami parameter of the former crystallization was determined to be 1.23 ± .12, which indicated two-dimensional crystal growth with heterogeneous nucleation. The toxicity characteristic leaching procedure results indicated that the heavy metals were well solidified, and that the leaching concentration was significantly lower than the limit specified by governmental agencies. The potentially toxic element index of the parent glass and the two glass ceramics were 11.7, 5.8, and 3.6, respectively. Therefore, the conversion of hazardous petrochemical incineration fly ash and other solid waste into environmentally friendly glass ceramics shows considerable potential and reliability.

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis.

OBJECTIVE: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology. METHODS: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre+ UVRAGfl/fl (M-UVRAG-/-) mice were compared to littermate Myf6-Cre+ UVRAG+/+ (M-UVRAG+/+) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age. RESULTS: M-UVRAG-/- mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG+/+ littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG-/- mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis. CONCLUSIONS: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.

γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K-dependent proteins.

γ-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K-dependent (VKD) proteins involved in a wide variety of physiologic processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype-phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of 3 structure-function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter proteins, and then the carboxylation efficiency of these reporter proteins was evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C terminus plays a critical role in the binding of osteocalcin but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein-protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre-messenger RNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes and clarified why vitamin K administration rectified bleeding disorders but not nonbleeding disorders.

OIP5-AS1 specifies p53-driven POX transcription regulated by TRPC6 in glioma.

Transcription factors (TFs) control an array of expressed genes. However, the specifics of how a gene is expressed in time and space as controlled by a TF remain largely unknown. Here, in TRPC6-regulated proline oxidase 1 (POX) transcription in human glioma, we report that OIP5-AS1, a long noncoding RNA, determines the specificity of p53-driven POX expression. The OIP5-AS1/p53 complex via its 24 nucleotides binds to the POX promoter and is necessary for POX expression but not for p21 transcription. An O-site in the POX promoter to which OIP5-AS1 binds was identified that is required for OIP5-AS1/p53 binding and POX transcription. Blocking OIP5-AS1 binding to the O-site inhibits POX transcription and promotes glioma development. Thus, the OIP5-AS1/O-site module decides p53-controlled POX expression as regulated by TRPC6 and affects glioma development.

Reevaluation of the contributions of reactive intermediates to the photochemical transformation of 17ß-estradiol in sewage effluent.

Photodegradation of the natural steroid 17ß-estradiol (E2), an endocrine-disrupting hormone that has been widely detected in aquatic environments, was investigated in wastewater effluents at various pH ranges under simulated solar irradiation. The rate of E2 degradation in the sewage effluents was stable at pH 6.0-7.0 but suddenly increased from pH 8.0-10.0. The second-order reaction rate constants of E2 with 3EfOM* and CO3•- were measured to increase 11.0-fold and 18.0-fold from pH 6.0 to 10.0, respectively. Two main reasons are proposed for this sharp increase. First, the change in the ionization state of E2 made it susceptible to oxidation by triplet-state effluent organic matter (3EfOM*) and carbonate radicals (CO3•-). Second, the steady-state concentration of CO3•- increased with increasing pH. Indirect photolysis was suggested to be the main degradation pathway in the sewage effluents, and 3EfOM* was proposed to play a major role at pH 8.0-9.0, while CO3•- played a significant role at pH 10.0. In this study, EfOM was shown for the first time to inhibit the oxidation of E2 initiated by 3EfOM* and CO3•-. Thus, we suggest that EfOM plays a dual role in the photodegradation of E2: EfOM can not only be activated as 3EfOM* to degrade E2 but also can inhibit the degradation of E2 by reducing the E2 oxidation intermediate back to E2. The estrogenic activity of the photodegradation products was also studied. The in vitro estrogenic activity of E2 solutions decreased approximately as fast as the E2 photodegradation occurred in the effluent water at various pH values, suggesting that solar photodegradation in sewage effluents reduces the risk of endocrine disruption in waters impacted by E2 and subject to continuing inputs. The results of this study are important for predicting the environmental fate of endocrine-disrupting chemicals and developing methods for their removal from aquatic environments.

Carbonate Radical Oxidation of Cylindrospermopsin (Cyanotoxin): Kinetic Studies and Mechanistic Consideration.

Cylindrospermopsin (CYN) is one of the most important cyanobacterial toxins frequently found in surface waters. We reported the detailed kinetics and pathways for the reaction of CYN with carbonate radicals (CO3•-). The rate constants of neutral and deprotonated CYN with CO3•- were found to be (1.2 ± 0.7) × 107 M-1 s-1 and (3.0 ± 0.4) × 108 M-1 s-1, respectively. The transformation products for the oxidation of CYN by CO3•- were identified by high-resolution mass spectrometry, illustrating that the guanidine and bridged hydroxyl portions were the primary moieties attacked by CO3•-. Thus, three transformation pathways, including cleavage of the hydroxymethyluracil moiety, hydroxylation, and oxidation of the bridged hydroxyl group, are proposed for the CO3•- oxidation of CYN. Moreover, this study reported that dissolved organic matter (DOM) reduced the transformation rate of CYN by inhibiting the transformation of oxidation intermediates. Finally, the role of CO3•- in CYN degradation was estimated in both sunlit surface waters and advanced oxidation processes (AOPs), demonstrating that CO3•- played an important role in CYN attenuation under nonacidic environmentally relevant conditions. The kinetic parameters and product information obtained in this study will be of considerable interest for the application of AOPs and predicting the environmental fate of CYN.

A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle.

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study.

Vitamin K-dependent carboxylation is a post-translational modification essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With the discovery of new vitamin K-dependent proteins, the importance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor's propeptide controls substrate binding and product releasing during carboxylation, and the propeptide of factor IX appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin's hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocalcin cannot direct the carboxylation of the coagulation factor, it is required for the efficient carboxylation of osteocalcin. This suggests that the coagulation factors may have a different mechanism of carboxylation from osteocalcin. Together, results from this study provide insight into efficiently controlling one physiological process, such as coagulation without affecting the other, like bone metabolism.

A Novel and Fast Purification Method for Nucleoside Transporters.

Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.

Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River.

Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.

Genome Sequence of a Typical Ultramicrobacterium, Curvibacter sp. Strain PAE-UM, Capable of Phthalate Ester Degradation.

Curvibacter sp. strain PAE-UM, isolated from river sediment, is a typical ultramicrobacterium capable of phthalate ester degradation. The genome of Curvibacter sp. PAE-UM consists of 3,284,473 bp, and its information will provide insights into the molecular mechanisms underlying its degradation ability.

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Genome Sequence of a Freshwater Low-Nucleic-Acid-Content Bacterium, Betaproteobacterium Strain CB.

Betaproteobacterium strain CB is a typical minute freshwater bacterium, representing the small-cell bacteria that are numerically dominant in most freshwater environments. The genome of betaproteobacterium CB consists of a circular 2,045,720-bp chromosome, and the information we report will provide insights into the mechanisms underlying its survival and ecological function.

Characterization of a novel serine protease inhibitor gene from a marine metagenome.

A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with K(i) values of around 1.79 × 10(-8) and 1.52 × 10(-8) M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0-8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.