90-kDa Protein Research Articles

Metallo-supramolecular complexes enantioselectively target monkeypox virus RNA G-quadruplex and bolster immune responses against MPXV

Abstract Mpox viruses (MPXV) has emerged as a formidable orthopoxvirus, posing an immense challenge to global public health. Understanding the regulatory mechanisms of MPXV infection, replication and immune evasion will benefit the development of novel antiviral strategies. Despite the involvement of G-quadruplexes (G4s) in modulating infection and replication processes of multiple viruses, their roles in MPXV life cycle remain largely unknown. Here, we found a highly conservative and stable G4 in MPXV, which acts as a positive regulatory element for viral immunodominant protein expression. Furthermore, by screening 42 optically pure chiral metal complexes, we identified the Λ enantiomer of a pair of chiral helical compounds can selectively target mRNA G4 and enhance expression of the 39-kDa core protein encoded by the MPXV A5L gene. Mechanistically, RNA G4-specific helicase DHX36 inhibits A5L protein expression by unwinding G4. In contrast, MH3 Λ enhanced mRNA stability by specifically targeting G4 structures and subsequently increased protein expression. Furthermore, given the pivotal role of the 39-kDa core protein in activating immune responses and facilitating virion maturation, modulation of MPXV G4 folding by MH3 Λ exhibited inhibitory effects on MPXV replication through enhancing immune response. Our findings underscore the critical involvement of G4 in MPXV life cycle and offer potential avenues for developing antiviral drugs targeting G4.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Self-assembled superstructure alleviates air-water interface effect in cryo-EM

Cryo-electron microscopy (cryo-EM) has been widely used to reveal the structures of proteins at atomic resolution. One key challenge is that almost all proteins are predominantly adsorbed to the air-water interface during standard cryo-EM specimen preparation. The interaction of proteins with air-water interface will significantly impede the success of reconstruction and achievable resolution. Here, we highlight the critical role of impenetrable surfactant monolayers in passivating the air-water interface problems, and develop a robust effective method for high-resolution cryo-EM analysis, by using the superstructure GSAMs which comprises surfactant self-assembled monolayers (SAMs) and graphene membrane. The GSAMs works well in enriching the orientations and improving particle utilization ratio of multiple proteins, facilitating the 3.3-Å resolution reconstruction of a 100-kDa protein complex (ACE2-RBD), which shows strong preferential orientation using traditional specimen preparation protocol. Additionally, we demonstrate that GSAMs enables the successful determinations of small proteins (<100 kDa) at near-atomic resolution. This study expands the understanding of SAMs and provides a key to better control the interaction of protein with air-water interface.

Morphometric analysis of spinal motor neuron degeneration in sporadic amyotrophic lateral sclerosis

ObjectivesThis study aimed to clarify the relationship between 43-kDa TAR DNA-binding protein (TDP-43) pathology and spinal cord anterior horn motor neuron (AHMN) atrophy in sporadic amyotrophic lateral sclerosis (SALS). MethodsEight patients with SALS and 12 controls were included in this study. Formalin-fixed specimens of lumbar spinal cord samples were paraffin-embedded and sectioned at the level of the fourth lumbar spinal cord with a 4 μm thickness. Using a microscope, the long diameters of the neurons with nucleoli were measured in spinal AHMNs stained with an anti-SMI-32 antibody. AHMNs were divided into medial and lateral nuclei for statistical analysis. We also used previously reported data to measure the long diameter of AHMNs with initial TDP-43 pathology, in which TDP-43 was present both in the nucleus and cytoplasm. ResultsThe long diameter of the lumbar spinal AHMNs in patients with SALS was smaller in the medial nucleus (42.54 ± 9.33 μm, n = 24) and the lateral nucleus (49.41 ± 13.86 μm, n = 129) than in controls (medial nucleus: 55.84 ± 13.49 μm, n = 85, p < 0.001; lateral nucleus: 62.39 ± 13.29 μm, n = 756, p < 0.001, Mann–Whitney U test). All 21 motor neurons with initial TDP-43 pathology were in the lateral nucleus, and their long diameter (67.60 ± 18.3 μm, p = 0.352) was not significantly different from that of controls. ConclusionMotor neuron atrophy in SALS does not occur during the initial stages of TDP-43 pathology, and TDP-43 pathology is already advanced in the atrophied motor neurons.

Structure and function of vasa gene in gonadal gametogenesis of Pacific abalone

Pacific abalone (Haliotis discus hannai) is a marine gastropod mollusc with significant economic importance in both global fisheries and aquaculture. However, studies exploring the gonadal development and regulatory mechanisms of Haliotis discus hannai are limited. This study aimed to explore whether the vasa gene acted as a molecular marker for germ cells. Initially, the vasa gene was successfully cloned using the cDNA-end rapid amplification technique. The cloned gene had a 2478-bp-long open reading frame and encoded 825 amino acids. Then, a recombinant expression vector was constructed based on the Vasa protein, and an 87-kDa recombinant protein was prepared. Subsequently, a polyclonal antibody was prepared using the purified recombinant protein. The enzyme-linked immunosorbent assay (ELISA) confirmed the titer of the antibody to be ≥512 K. The immunohistochemical analysis revealed that Vasa was widely expressed in oogonia, Stage I oocytes, spermatogonia, and primary spermatocytes. The specific expression of Vasa in the hermaphroditic gonads of abalone was assessed using western blotting to investigate the effects of different photoperiods (12 L:12D, 24 L:0D, 18 L:6D, and 6 L:18D) on the gonadal development of abalone (P < 0.05), with higher expression levels observed in the ovarian proliferative and spermary maturing stages compared with other developmental stages (P < 0.05). Additionally, Vasa exhibited the highest expression in the spermary and ovary under a photoperiod of 18 L:6D (P < 0.05). These data demonstrated the key role of Vasa in developing germ cells in abalone. They shed light upon the molecular mechanism through which the photoperiod influenced Vasa expression and regulated gonadal development in abalone. The findings might provide theoretical references for analyzing the differentiation pattern of abalone germ cells and the genetic improvement and conservation of germplasm resources.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E . coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Genotyping of ticks: first molecular report of Hyalomma asiaticum and molecular detection of tick-borne bacteria in ticks and blood from Khyber Pakhtunkhwa, Pakistan.

Multiple ticks (Acari: Ixodoidea) carrying Rickettsiales bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus, Rhipicephalus microplus, Haemaphysalis cornupunctata, and Hyalomma asiaticum. Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase (cox1) gene. Obtained cox1 sequences of four different tick species showed 99.72%-100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA, 190-kDa outer membrane protein A (ompA), and 120-kDa outer membrane protein B (ompB) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle's blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%-99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle's blood and ticks (Hae. cornupunctata) showed 99.67% highest identity with Anaplasma phagocytophilum. The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities.

Investigation of periodontal disease development and Porphyromonas gulae FimA genotype distribution in small dogs

In dogs, Porphyromonas gulae is a major periodontal pathogen with 41-kDa proteins polymerizing to form a filamentous structure called fimbriae or pili, termed FimA. FimA is classified into three genotypes: A, B, and C, and there are combinations of types A, B, C, A/B, A/C, B/C, and A/B/C. Periodontal disease is the most common oral disease in small dogs, but the periodontal disease status and P. gulae colonization at each dog age and breed remain unclear. In this study, we stratified 665 small dogs and analyzed the periodontal status and distribution of P. gulae with each FimA genotype. Dogs with periodontal disease and FimA genotype tended to increase with age. The dogs with at least one FimA genotype had significantly more severe periodontal disease compared with P. gulae-negative dogs (P < 0.01). Additionally, periodontal status was significantly associated with specific FimA genotype distribution in Toy Poodles and Chihuahuas (P < 0.05), whereas there was no such association in Dachshunds. These results suggest that the onset of periodontal disease and P. gulae colonization are related and progress with age. The relationship between periodontal disease and FimA genotype may differ depending on the dog breeds.

Cryo-EM structure of a 55-kDa nucleoplasmin protein

Cryo-EM structure of a 55-kDa nucleoplasmin protein

Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.

The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.

Bacillus thuringiensis Bt_UNVM-84, a Novel Strain Showing Insecticidal Activity against Anthonomus grandis Boheman (Coleoptera: Curculionidae).

Bacillus thuringiensis is a Gram-positive bacterium known for its insecticidal proteins effective against various insect pests. However, limited strains and proteins target coleopteran pests like Anthonomous grandis Boheman, causing substantial economic losses in the cotton industry. This study focuses on characterizing a Bacillus sp. strain, isolated from Oncativo (Argentina), which exhibits ovoid to amorphous parasporal crystals and was designated Bt_UNVM-84. Its genome encodes genes for the production of two pairs of binary Vpb1/Vpa2 proteins and three Cry-like proteins showing similarity with different Cry8 proteins. Interestingly, this gene content was found to be conserved in a previously characterized Argentine isolate of B. thuringiensis designated INTA Fr7-4. SDS-PAGE analysis revealed a major band of 130 kDa that is proteolytically processed to an approximately 66-kDa protein fragment by trypsin. Bioassays performed with spore-crystal mixtures demonstrated an interesting insecticidal activity against the cotton boll weevil A. grandis neonate larvae, resulting in 91% mortality. Strain Bt_UNVM-84 is, therefore, an interesting candidate for the efficient biological control of this species, causing significant economic losses in the cotton industry in the Americas.

Effect of the ati Gene Deletion on the Pathogenicity and Immunogenicity of the Vaccinia Virus.

Among the nonvirion proteins of the vaccinia virus (VACV), a 94-kDa long protein is most abundantly present; the protein is a truncated form of the 150-kDa A-type inclusion (ATI) protein of the cowpox virus encoded by the ati gene. This VACV protein does not form intracellular ATIs, being as it is a major immunogen upon infection/immunization of humans or animals with the VACV. Antibodies specific to this protein are not virus-neutralizing. The present study focused on the effect of the production of this nonstructural major immunogenic VACV protein on the manifestation of pathogenicity and immunogenicity of the virus in the BALB/c mouse model of infection. In order to introduce a targeted deletion into the VACV LIVP genome, the recombinant integration/deletion plasmid pΔati was constructed and further used to generate the recombinant virus LIVPΔati. The pathogenicity of the VACV LIVP and LIVPΔati strains was studied in 3-week-old mice. The mice were intranasally infected with the viruses at a dose of 107 pfu; 50% of the animals infected with the parent LIVP strain died, while infection with the LIVPΔati strain led to the death of only 20% of the mice. Intradermal vaccination of mice aged 6- weeks with the LIVPΔati virus statistically significantly increased the production of VACV-specific IgG, compared to that after intradermal vaccination with VACV LIVP. Meanwhile, no differences were noted in the cell-mediated immune response to the vaccination of mice with VACV LIVP or LIVPΔati, which was assessed by ELISpot according to the number of splenocytes producing IFN-γ in response to stimulation with virus-specific peptides. Intranasal infection of mice with lethal doses of the cowpox virus or the ectromelia virus on day 60 post-immunization with the studied VACV variants demonstrated that the mutant LIVPΔati elicits a stronger protective response compared to the parent LIVP.

Polypyrimidine Tract Binding Protein: A Universal Player in Cancer Development.

Polypyrimidine tract binding protein is a 57-Kda protein located in the perinucleolar compartment where it binds RNA and regulates several biological functions through the regulation of RNA splicing. Numerous research articles have been published that address the cellular network and functions of PTB and its isoforms in various disease states. Through an extensive PubMed search, we attempt to summarize the relevant research into this biomolecule. Besides its roles in embryonic development, neuronal cell growth, RNA metabolism, apoptosis, and hematopoiesis, PTB can affect cancer growth via several metabolic, proliferative, and structural mechanisms. PTB overexpression has been documented in several cancers where it plays a role as a novel prognostic factor. The diverse carcinogenic effect opens an argument into its potential role in inhibitory targeted therapy.

Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment seven (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment seven RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the ~120-kDa S1 protein. The ~120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of ~80 kDa. Co-pulldown assays demonstrated that the ~120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples

Treatment at the early stage of onset is vital for the prognosis of rickettsioses. But the absence of specific clinical symptoms complicates the diagnosis of this condition. Herein we established a seminested recombinase polymerase amplification assay (snRPA-nfo) that enables quick detection and differentiation of rickettsial pathogens in clinical samples with high sensitivity and specificity. The conserved 17-kDa protein gene of Rickettsia sibirica and the 47-kDa protein gene of Orientia tsutsugamushi were targeted for the duplex RPA-nfo assay. The snRPA-nfo assay exhibited an increased LOD in spiked blood samples, up to 1000-fold in comparison to standard RPA-nfo, and a better detection rate (83.3%, 5/6) than TaqMan PCR (16.6%, 1/6, Ct ≤ 35) in clinically confirmed patient blood samples. Thus, snRPA-nfo assay represents a promising alternative to TaqMan PCR in the early diagnosis of rickettsioses for point-of-care testing as well as in resource-limited settings.

The Evolution of Polyclonal Antibody from Specific Epitope 47kDA for Detection of Bladder Cancer.

This study will identify specific epitopes from the 47kDa protein as the basis for making polyclonal antibodies to increasing sensitivity and specificity of 47kDa protein as bladder cancer biomarkers. The 47kDa protein epitope prediction was carried out using the in-silico method. The epitope with the highest and the lowest value was immunized to the mice for four weeks and was harvested at the fifth weeks. The antibody was tested with the patient's urine using western blotting. Total of 186 participants including in this study. For the first stage (antibody confirmation test) test we have 18 participants, for the second stage (1st antibody diagnosis test) we have 72 participants and for the third stage (2nd antibody diagnosis test) we have 96 participants, consist of total 64 BC patients 48 of healthy individuals and 74 participants with the other diseases. Some epitopes from the sequenced protein are candidates for immunization, in the chain 108'-136' (with lowest Bepipred score: 0.53) named as peptide1 and chain 42'-56' (with highest bepipred score: 0.58) named as peptide2. In western blotting test, both antibodies showed detection at 47kDa. When examined with western blot using urine from BC patients, urine from other cancer patients (prostate, kidney, ureter, rectal, breast), and healthy persons, both antibodies were found to only express 47kDa in urine from BC patients. The diagnostic tests showed high sensitivity (91.67%) and specificity (94.44%) inAb2 in predicting bladder cancer. The evolution of the polyclonal antibody made from specific epitopes is proven to express specifically on bladder cancer patients and have high sensitivity and specificity to diagnose bladder cancer.

Evaluation of haemolymph phenoloxidase activity from the grub of Zophobas morio as a predictor of immune response.

In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30°C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85kDa and 55kDa polypeptides in all the experiments over control after 24h, 48h and 96h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55kDa protein and 85kDa protein. A significant increase in 85kDa polypeptide was observed in the haemolymph of the grubs after 72h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.

Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125kDa) and X2 variants (88 and 79kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.