Uncovering novel drug targets for bipolar disorder: a Mendelian randomization analysis of brain, cerebrospinal fluid, and plasma proteomes.

BACKGROUND: There is a clear demand for innovative therapeutics for bipolar disorder (BD). METHODS: We integrated the largest BD genome-wide association study (GWAS) dataset (NCase = 41 917, NControl = 371 549) with protein quantitative trait loci from brain, cerebrospinal fluid, and plasma. Using a range of integrative analyses, including Mendelian randomization (MR), Steiger filter analysis, Bayesian colocalization, and phenome-wide MR analysis, we prioritized novel drug targets for BD. Additionally, we incorporated data from the UK Biobank (NCase = 1064, NControl = 365 476) and the FinnGen study (NCase = 7006, NControl = 329 192) for robust biological validation. RESULTS: Through MR analysis, we found that in the brain, downregulation of DNM3, MCTP1, ABCB8 and elevation of DFNA5 and PDF were risk factors for BD. In cerebrospinal fluid, increased BD risk was associated with increased levels of FRZB, AGRP, and IL36A and decreased CTSF and LRP8. Plasma analysis revealed that decreased LMAN2L, CX3CL1, PI3, NCAM1, and TIMP4 correlated with increased BD risk, but ITIH1 did not. All these proteins passed Steiger filtering, and Bayesian colocalization confirmed that 12 proteins were colocalized with BD. Phenome-wide MR analysis revealed no significant side effects for potential drug targets, except for LRP8. External validation further underscored the concordance between the primary and validation cohorts, confirming MCTP1, DNM3, PDF, CTSF, AGRP, FRZB, LMAN2L, NCAM1, and TIMP4 are intriguing targets for BD. CONCLUSIONS: Our study identified druggable proteins for BD, including MCTP1, DNM3, and PDF in the brain; CTSF, AGRP, and FRZB in cerebrospinal fluid; and LMAN2L, NCAM1, and TIMP4 in plasma, delineating promising avenues to development of novel therapies.

Abnormal Brain Protein Abundance and Cross-tissue mRNA Expression in Amyotrophic Lateral Sclerosis.

Due to the limitations of the present risk genes in understanding the etiology of amyotrophic lateral sclerosis (ALS), it is necessary to find additional causative genes utilizing novel approaches. In this study, we conducted a two-stage proteome-wide association study (PWAS) using ALS genome-wide association study (GWAS) data (N = 152,268) and two distinct human brain protein quantitative trait loci (pQTL) datasets (ROSMAP N = 376 and Banner N = 152) to identify ALS risk genes and prioritized candidate genes with Mendelian randomization (MR) and Bayesian colocalization analysis. Next, we verified the aberrant expression of risk genes in multiple tissues, including lower motor neurons, skeletal muscle, and whole blood. Six ALS risk genes (SCFD1, SARM1, TMEM175, BCS1L, WIPI2, and DHRS11) were found during the PWAS discovery phase, and SARM1 and BCS1L were confirmed during the validation phase. The following MR (p = 2.10 × 10-7) and Bayesian colocalization analysis (ROSMAP PP4 = 0.999, Banner PP4 = 0.999) confirmed the causal association between SARM1 and ALS. Further differential expression analysis revealed that SARM1 was markedly downregulated in lower motor neurons (p = 7.64 × 10-3), skeletal muscle (p = 9.34 × 10-3), and whole blood (p = 1.94 × 10-3). Our findings identified some promising protein candidates for future investigation as therapeutic targets. The dysregulation of SARM1 in multiple tissues provides a new way to explain ALS pathology.

A Novel Form of Arginine-Chitosan as Nanoparticles Efficient for siRNA Delivery into Mouse Leukemia Cells.

The modification of chitosan (CS) has greatly expanded its application in the field of medicine. In this study, low-molecular-weight chitosan was modified with arginine (Arg) by a simple method. The identification by the Fourier transform infrared spectra (FTIR) showed that Arg was successfully covalently attached to the CS. Interestingly, Arg-CS was identified as nanoparticles by atomic force microscopy (AFM) and transmission electron microscopy (TEM), whose particle size was 75.76 ± 12.07 nm based on Dynamic Light Scattering (DLS) characterization. Then, whether the prepared Arg-CS nanoparticles could encapsulate and deliver siRNA safely was investigated. Arg-CS was found to be able to encapsulate siRNAs in vitro via electrostatic interaction with siRNA; the Arg-CS/siRNA complex was safe for L1210 leukemia cells. Therefore, modification of chitosan by Arg produces novel nanoparticles to deliver siRNA into leukemia cells. This is the first time to identify Arg-CS as nanoparticles and explore their ability to deliver Rhoa siRNA into T-cell acute lymphoblastic leukemia (T-ALL) cells to advance therapies targeting Rhoa in the future.

Exploring of seasonal dynamics of microbial community in multispecies fermentation of Shanxi mature vinegar.

The constituents of fermentation foods vary seasonally and the microbiota plays a crucial role in metabolites formation. Here, the diversity and succession of microbiota of Shanxi mature vinegar produced with solid-solid fermentation craft have been investigated by Illumina Hiseq sequencing in both summer and winter. Obvious differences were observed in the structure of microbiota between summer and winter, and the bacterial community showed a significant difference (P < 0.05). Alpha diversity analysis showed that the diversity and richness of bacterial community were basically higher than that of fungal community in both summer and winter. For bacterial community, Lactobacillus and Limosilactobacillus were the two major group bacteria in the fermentation process of Shanxi mature vinegar in summer, and they dominated in acetic acid fermentation and alcoholic fermentation stages, respectively. Lactobacillus and Acetobacter were the two major group bacteria during the fermentation of Shanxi mature vinegar in winter. Saccharomyces, Saccharomycopsis, and Issatchenkia were the main yeasts in both seasons, while the dominant mould was Rhizopus in summer and Monascus in winter, respectively. The diversity of yeasts and moulds in winter was far greater than that in summer, especially in alcoholic fermentation stage. Collectively, our work revealed critical insights into effect of seasonal variation on the structure of microbiota of Shanxi mature vinegar, and was relevant in understanding the relationships between environmental change and microbiota.

Enhancement of Antibacterial and Mechanical Properties of Photocurable ε-Poly-l-lysine Hydrogels by Tannic Acid Treatment.

In this study, a photocurable hydrogel based on an ε-poly-l-lysine (EPL) composite was fabricated by a grafting reaction using glycidyl methacrylate and then complexed with tannic acid (TA) to improve the mechanical stability and antibacterial performance of the EPL hydrogels. UV-visible spectrophotometry, nuclear magnetic resonance, and Fourier transform infrared spectroscopy were introduced to characterize the chemical construction. The obtained EPLMA hydrogel was immersed into TA solution to induce the forming of the H-bond between EPL and TA, resulting in double networks in the composite hydrogel (EPLMA-TA). Due to the additional hydrogen-bond interaction between TA and EPLMA, the mechanical properties of hydrogels were improved and supported cell growth and proliferation. In addition, the antibacterial properties and antioxidant activities of the EPLMA-TA hydrogels were greatly enhanced due to the addition of TA. All the findings indicate that the EPLMA-TA hydrogels with multiple properties show great potential for biomedicine applications.

Draft Genome Sequence of Rummeliibacillus sp. Strain TYF005, a Physiologically Recalcitrant Bacterium with High Ethanol and Salt Tolerance Isolated from Spoilage Vinegar.

Rummeliibacillus sp. strain TYF005 is a thermophilic bacterium with high ethanol (8% vol/vol) and salt (13% wt/vol) tolerance that was isolated from spoilage vinegar. Here, we report the draft genome sequence of this strain, which has 117 scaffolds with a total genome size of 3.7 Mb and a 34.4% GC content.

Secretory expression, immunoaffinity purification and metal-binding ability of recombinant metallothionein (ShMT) from freshwater crab Sinopotamon henanense.

Metallothioneins (MTs) are a super-family of ubiquitous, low-molecular-weight, cysteine-rich and metal-binding proteins. They are thought to play a predominant role in mediating metal metabolism and antioxidation. However, the accurate functions of MTs remain unclear in the physiological processes due to native proteins deficiency and little information of their metal-binding character. Freshwater crab Sinopotamon henanense is a decapod crustacean widely distributed in northern China, in which only one MT isoform (ShMT) has been reported so far. In order to shed light on the accurate role of ShMT, a novel recombinant ShMT in native form was over-expressed by phoA secreted expression system in Escherichia coli (E. coli). Then the ShMT proteins were purified using a one-step gentle immunoaffinity chromatography with a polyol-responsive mAb (PR-mAb) to ShMT, which was generated by conventional hybridoma technology followed by ELISA-elution. The Zn-, Cu-, and Cd-ShMT complexes were prepared by recombinant synthesis in metal-enriched media and reconstitution with metal ions, respectively. Further analysis about metal-binding capacity showed recombinant ShMT has high ability to bind Zn, Cu and Cd metals, although the recombinantly expressed and reconstituted metal-ShMT complexes have different metal-to-protein stoichiometry. Moreover, the affinity of recombinant protein for metal ions has been analyzed using competitive reaction with 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB). The results demonstrated the affinity of recombinant ShMT for metals was as follows: Cu＞Cd＞Zn. In summary, the experimental procedure we have developed facilitates production of recombinant ShMT with native characteristics for further research and the study of metal-binding ability could help further clarify the accurate functions of ShMT.

[Preparation and characterization of a monoclonal antibody against human tissue factor with anticoagulation activity].

OBJECTIVE: To prepare and characterize a monoclonal antibody (mAb) against human tissue factor (hTF) with anticoagulation activity. METHODS: BALB/c mice were immunized with truncated recombinant protein (rhTF243). Hybridoma cell lines were generated from cell fusion, and screened using indirect ELISA and prothrombin time (PT). After ascites was developed in BALB/c mice, antibody titers were determined using indirect ELISA. Western blotting was performed to study the antibody specificity. Anticoagulant activity of the antibody was detected by PT assay. RESULTS: A mAb to hTF with excellent anticoagulation activity was identified. Its immunoglobulin subclass belonged to IgG1. Titer of ascites fluid was 1:200 000. Western blotting and PT analysis confirmed the specificity and anticoagulant activity of the antibody. The mAb reacted specifically to both recombinant hTF243 and natural TF on SW620 colon cancer cell surface. CONCLUSION: A hTF mAb with anticoagulation activity and high specificity has been successfully prepared.

Subcellular distribution of Cd and Zn and MT mRNA expression in the hepatopancreas of Sinopotamon henanense after single and co-exposure to Cd and Zn.

The freshwater crab Sinopotamon henanense was exposed to Cd and Zn either on their own or in combination. At 14 and 28 days the hepatopancreas was taken and total metal and metallothionein MT) in transcript abundance were assessed. In addition, the subcellular contents of Cd and Zn also were examined following a differential centrifugation. The results showed that concentration of Cd was more responsive to waterborne metal exposures than the concentration of Zn; Zn was better regulated and exhibited only 2-3 fold increases relative to the control. Of the subcellular fractions, the heat stable protein (HSP) fraction was the predominant metal-binding compartment for Cd. The proportion and accumulation of Cd in this fraction increased with the single Cd exposures, which suggest that metallothionein-like proteins play a key role in metal detoxification in the hepatopancreas of S. henanense. Despite the increases in Cd in the HSP fraction during single Cd exposures, some accumulation of Cd was observed in metal sensitive fraction (MSF), which showed that metal detoxification was incomplete. The results demonstrated that the Cd content decreased in metal mixture groups especially when mixed with higher Zn, while the Cd accumulation in MSF was also reduced when Zn was added. MT mRNA expression was also determined in our report, the results showed that both Cd and Zn had the ability of inducing MT mRNA expression. Additionally, the MT mRNA transcription level was enhanced when Cd was mixed with Zn.

Mitochondrial energy metabolism in the hepatopancreas of freshwater crabs (Sinopotamon henanense) after cadmium exposure.

Energetic homoeostasis is a fundamental requirement in the defense against cadmium (Cd) stress. Using the freshwater crab Sinopotamon henanense as an example, we explored the effects of Cd on submicroscopic structures in hepatopancreatic cells through transmission electron microscopy (TEM) and energy-related parameters such as adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratios, reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD(+)) ratios, and the mitochondrial membrane potential (Δψm). The impact of Cd on carbohydrate and protein metabolism, and metallothionein (MT) was also investigated. Experimental 10 day exposure increased NADH and NAD(+) and provided a higher NADH/NAD(+) ratio and Δψm in hepatopancreatic cells. The corresponding significantly increased levels of ATP and the ATP/ADP ratio in the hepatopancreas supported high energy production. The up-regulation of the MT content in exposed crabs suggests that protein synthesis for detoxification could partially be a major ATP consumer. With increasing exposure time, however, energy production was in decline. Excessive energy consumption was explained by substrate mobilization and mitochondrial impairment. Less carbohydrate and enhanced protein catabolism was observed. Ultrastructurally, there were changes in mitochondria with swelling, membrane disruption, shortening of cristae or the rupture and disappearance of entire mitochondria. The rough endoplasmic reticulum (rER) displayed expansion and membrane rupture, suggesting the destruction of protein-synthesizing structures in hepatopancreatic cells. Our findings suggest that energy-related parameters could be used as biomarkers in the monitoring of metal pollution and quantitative risk assessments of pollutant exposure.

[Preparation and characterization of monoclonal antibodies against metallothionein of freshwater crab (Sinopotamon henanense)].

OBJECTIVE: To prepare a monoclonal antibody (mAb) against metallothionein (MT) of freshwater crab (Sinopotamon henanense) and characterize its immunologic properties. METHODS: Two recombinant MT of S.henanense, namely SUMO-MT and His-MT, were produced by SUMO fusion system and phoA secretion expression system in E.coli. SUMO-MT was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning, repeated screening with His-MT as detecting antigen, the hybridomas specifically secreting mouse mAb against the MT of S. henanense were generated. The titers of mAbs were measured by indirect ELISA and the specificity of the mAbs was evaluated by Western blotting and Dot-ELISA. RESULTS: Two hybridoma cell lines designated mAb-MT2 and mAb-MT3 with the property of secreting mAb against the MT continuously and steadily were successfully obtained. Their immunoglobulin subclass was IgG1. The titers of the ascites fluid were 1:500 000 and 1:1000 000, respectively. Western blot analysis confirmed that the two mAbs both reacted with recombinant SUMO-MT and His-MT with good sensitivity. The Dot-ELISA demonstrated that the two mAbs reacted specifically not only with recombinant MT but also natural MT. CONCLUSION: The mAbs against MT of S. henanense with high specificity were successfully prepared.

Tissue-specific copper accumulation, zinc levels, induction, and purification of metallothionein in freshwater crab Sinopotamon henanense exposed to subacute waterborne copper.

Copper (Cu) is one of the most important essential metals for crustaceans, buttoxic in excess. Metallothioneins (MT) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis, detoxification, and cytoprotection. In the present study, Sinopotamon henanense were exposed to 0 (controls), 2.86, and 14.3 mg L(-1) waterborne Cu, Cu accumulation, zinc (Zn) levels and MT induction in gills and hepatopancreas were determined with Cd/Hemoglobin saturation assay and atomic absorption spectrophotometry method. Results showed that Cu accumulation and MT levels were both tissue-specific and revealed some time-dependent and dose-dependent, respectively. The highest Cu accumulations of 82.10 ± 16.38 µg g(-1) w wt were observed in the gill after 15 days of 14.3 mg L(-1) Cu exposure, the peak MT induction of 136.16 ± 19.39 µg g(-1) w wt were observed in the hepatopancreas after 3 day of 14.3 mg L(-1) Cu exposure.In addition, the essential metal homeostasis of Zn was disturbed in some ways by subacute Cu exposure. The calculated ratios of actual Cu to theoretical maximum metal bound by MT indicating that the hepatopancreas had much greater Cu-binding potentials than the gills. Positive correlation were shown between MT induction and Cu accumulation both in hepatopancreas and gills, indicating that MT induction in S. henanense can be considered as a biomarker for subacute waterborne Cu pollution. Furthermore, the Cu induced MT (CuMT) from S. henanense was purified using acetone precipitation (50-80%), followed by gel filtration chromatography and anion exchange chromatography. SDS-PAGE and time-of-flight mass spectrometry analysis showed that S. henanense CuMT possess two isoforms and both mainly existed as monomer and dimmer forms. These present studies will be helpful to increase the database information of heavy metal-induced MT in terms of crustaceans.

Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium.

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.

Purification and cDNA cloning of a cadmium-binding metallothionein from the freshwater crab Sinopotamon henanense.

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are useful biomarkers for monitoring pollution by heavy metals. In this report, a novel cadmium (Cd)-binding MT (CdMT) from Sinopotamon henanense was purified using acetone precipitation (50-80%), followed by gel-filtration chromatography and anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and time-of-flight mass spectrometry analysis showed that S. henanense CdMT existed as monomer and dimmer forms, with a monomer molecular weight of 6890 Da and a dimmer molecular weight of 13,766 Da. In addition, the full-length cDNA sequence of S. henanense CdMT was prepared from the gill RNA using reverse transcription-polymerase chain reaction and 3' and 5' rapid amplification of cDNA ends (RACE) methods. Sequence analyses indicated that the isolated cDNA (633 bp) contains an open reading frame of 177 bp that encodes a protein with 59 amino acids. The deduced amino acid sequence has 18 cysteine residues, implying that S. henanense CdMT binds six equivalents of bivalent metal ions (Cd) as opposed to the seven in its mammalian counterparts. The deduced molecular weight of MT without binding metals is 6218 Da. If six bound Cd atoms are counted, the deduced molecular weight of S. henanense CdMT would be 6892 Da, which is very similar to the molecular weight of the purified protein (6890 Da) determined by time-of-flight mass spectrometry analysis. These confirmed our results of MT purification. These present studies will be helpful to increase the database information of heavy-metal-induced MT in terms of crustaceans.

Tissue-specific cadmium and metallothionein levels in freshwater crab Sinopotamon henanense during acute exposure to waterborne cadmium.

The freshwater crabs Sinopotamon henanense were exposed to different concentrations of waterborne cadmium (Cd). The relationship between tissue-specific Cd accumulation and metallothionein (MT) induction was investigated using the Cd saturation assay and atomic absorption spectrophotometry method. The results showed that Cd accumulation rose significantly in all tissues studied after Cd exposure, and the Cd accumulation level in various tissues followed the following order: gill > hepatopancreas > muscle > ovary. MT levels were clearly tissue-specific after Cd exposure. Hepatopancreas was found to have the highest MT level, followed by the gill, muscle, and ovary. In conclusion, the results indicated although Cd exposure clearly resulted in MT induction, its synthesis does not correlate with Cd accumulation in the later stage of Cd exposure. The calculated ratios of actual Cd to theoretical maximum Cd-MT in the hepatopancreas were <1.0 under acute waterborne Cd at all sampling points, indicating that the hepatopancreas had much greater Cd-binding potential of MT than the gill, muscle, or ovary. It is clear from our results that a positive correlation was shown between MT induction and Cd accumulation both in hepatopancreas and gill. Therefore, MT induction can be considered as a biomarker for acute waterborne Cd pollution.