In silico design of a novel hybrid epitope-based antigen harboring highly exposed immunogenic peptides of BamA, OmpA, and Omp34 against Acinetobacter baumannii.

Acinetobacter baumannii, is among the highest priority bacteria according to the WHO categorization which necessitate the exploration of alternative strategies such as vaccination. OmpA, BamA, and Omp34 are assigned as appropriate antigens to serve in vaccine development against this pathogen. Experimentally validated exposed epitopes of OmpA and Omp34 along with selected exposed epitopes predicted by an integrative in silico approach were represented by the barrel domain of BamA as a scaffold. Among the 8 external loops of BamA, 5 loops were replaced with selected loops of OmpA and Omp34. The designed antigen was analyzed regarding the physicochemical properties, antigenicity, epitope retrieval, topology, structure, and safety. BamA is a two-domain OMP with a 16-stranded barrel in which L4, L6, and L7 were the longest loops of BamA in order. The designed antigen consisted of 478 amino acids with antigen probability of 0.7793. The novel antigen was a 16-stranded barrel. No identical 8-meric peptides were found in the human proteome against the designed antigen sequence. The designed construct was safe regarding the allergenicity, toxicity, and human proteome reactivity. The designed antigen could develop higher protection against A. baumannii in comparison to either OmpA, BamA, or Omp34 alone.

Leptospira interrogans encodes a canonical BamA and three novel noNterm Omp85 outer membrane protein paralogs.

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three 'noNterm' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

Accumulation of heavy metals from single and combined olive mill wastewater and pomace in soil and bioaccumulation in tissues of two earthworm species: Endogeic (Aporrectodea trapezoides) and Epigeic (Eisenia fetida).

Soil and earthworms are threatened by anthropogenic contamination resulting from olive mill waste dumping on the soil due to their pollutant properties. While several studies have explored the effects of olive mill waste on soil properties and the accumulation of heavy metals in soil, there is currently a gap in the literature regarding the potential bioaccumulation of heavy metals from olive mill waste in earthworms. In this study, soil with earthworms from two ecological categories (endogeic: Aporrectodea trapezoides and epigeic: Eisenia fetida) was treated with increasing doses of olive mill wastewater (OMWW) and olive mill pomace (OMP), applied individually or combined, in an indoor experiment in plastic containers, under laboratory conditions. The results revealed the presence of significant concentrations of heavy metals in the two types of wastes ranging as follows: Fe˃ Zn˃ Cu˃ Cd˃ Cr for OMWW, and Fe˃ Zn˃ Cu˃ Cr for OMP (with Cd below the detection limit). The study demonstrated distinct effects of OMWW and OMP, both individually and in combination, on soil heavy metal content, ranging as follows: soil OMWW > soil Combination > soil OMP for Cd; soil Combination > soil OMWW > soil OMP for Cr and Fe; and soil Combination > soil OMP > soil OMWW for Cu and Zn. Additionally, our investigation showed that both earthworm species exhibited significant uptake of these metals into their tissues, particularly the endogeic species. Interestingly, the most significant difference between species was in the accumulation of Cu, with the epigeic species accumulating significantly lower amounts.

The chimeric UreB, FliD and Omp18 proteins for a sensitive and specific diagnosis of Helicobacter pylori infections.

Background and Objectives: Helicobacter pylori is known as the main cause of gastrointestinal diseases including gastritis, gastric ulcer and stomach cancer. Serodiagnosis of H. pylori infection is a noninvasive and rapid method but the efficiency of this method is highly dependent to the antigens used. This study evaluated the efficacy of recombinant UreB-Omp18 and FliD for serodiagnosis of H. pylori infection. Materials and Methods: The genes encoding for fliD, ureB, and omp18 was amplified by PCR and cloned into pET-22b and pET-28a vectors. The constructs were expressed in E. coli BL21 and purified by affinity chromatography. The antigenic properties and diagnostic potential of the recombinant proteins were analysed by immunoblotting and ELISA, respectively. Results: The recombinant UreB-Omp18 and FliD with molecular weights of 48 kDa and 25 kDa were observed on SDS-PAGE and purified by the Ni-NTA column. The ELISA results showed that the sensitivity and specificity of recombinant UreB-Omp18 protein in serodiagnosis of H. pylori infection were 89% and 83%, respectively. Also, the sensitivity and specificity of the recombinant FliD protein were calculated to be 91% and 76%, respectively. Conclusion: The results indicated that the recombinant UreB-Omp18 and FliD could diagnose H. pylori infection with high sensitivity and specificity.

Construction of recombinant Omp25 or EipB protein loaded PLGA nanovaccines for Brucellosis protection.

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed theirin vitro/in vivoimmunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to triggerBrucella-specific humoral and cellular immune response.

A comprehensive in silico analysis of putative outer membrane and secretory hydrolases from the pathogenic Leptospira: Possible implications in pathogenesis.

Outer surface/membrane and virulent secretory proteins are primarily crucial for pathogenesis. Secreted and outer membrane hydrolases of many pathogens play an important role in attenuating the host immune system. Leptospira expresses many such proteins, and few have been characterized to display various roles, including host immune evasion. However, identification, classification, characterization, and elucidation of the possible role of Leptospira's outer membrane and secretory hydrolases have yet to be explored. In the present study, we used bioinformatics tools to predict exported proteins from the pathogenic Leptospira proteome. Moreover, we focused on secretory and outer membrane putative hydrolases from the exported proteins to generate a deeper understanding. Our analysis yielded four putative outer/secretory hydrolases, LIC_10995, LIC_11183, LIC_11463, and LIC_12988, containing α/ß hydrolase fold and displayed similarity with lipase motif. Moreover, their conservation analysis of the predicted hydrolases across the spectrum of different Leptospira species showed high clustering with the pathogenic species. Outer membrane and secretory proteins with lipolytic activity may have a role in pathogenesis. This is the first bioinformatics analysis of secretory and outer membrane α/ß hydrolases from leptospiral species. However, experimental studies are indeed required to unravel this possibility.

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing.

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3-/4- on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Modelling competitive adsorption of organic micropollutants onto powdered activated carbon in continuous stirred tank reactors for advanced wastewater treatment.

This work investigates the validation and application of a competitive model approach for full-scale wastewater treatment plants (WWTP) with external recirculation of partially loaded powdered activated carbon (PAC) for removal of organic micropollutants (OMP). It is based on the ideal adsorbed solution theory (IAST) for multisolute mixtures combined with calibration of fictive organic components and correction of single-solute model parameters for OMP by use of the tracer model (TRM). Adsorption kinetics are represented by a pseudo first order reaction (PFO) and compared to mass transfer calculated with the homogenous surface diffusion model (HSDM). Model validation with operational data from two different WWTPs showed a strong dependency of model results on the batch sample quality used for model calibration. In contrast, the kinetic approach is of less importance for predicting full-scale OMP removal with long PAC sludge retention times. Further model application demonstrated that external PAC recirculation significantly improves the OMP removal with regard to both adsorption capacity and compensation of competitive effects of Dissolved Organic Carbon (DOC).

A team of chaperones play to win in the bacterial periplasm.

The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with ß-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.

An Improved OMP Algorithm for Enhancing the Anti-Interference Performance of Array Antennas.

The demand for precise positioning in noisy environments has propelled the development of research on array antenna radar systems. Although the orthogonal matching pursuit (OMP) algorithm demonstrates superior performance in signal reconstruction, its application efficacy in noisy settings faces challenges. Consequently, this paper introduces an innovative OMP algorithm, DTM_OMP_ICA (a dual-threshold mask OMP algorithm based on independent component analysis), which optimizes the OMP signal reconstruction framework by utilizing two different observation bases in conjunction with independent component analysis (ICA). By implementing a mean mask strategy, it effectively denoises signals received by array antennas in noisy environments. Simulation results reveal that compared to traditional OMP algorithms, the DTM_OMP_ICA algorithm shows significant advantages in noise suppression capability and algorithm stability. Under optimal conditions, this algorithm achieves a noise suppression rate of up to 96.8%, with its stability also reaching as high as 99%. Furthermore, DTM_OMP_ICA surpasses traditional denoising algorithms in practical denoising applications, proving its effectiveness in reconstructing array antenna signals in noisy settings. This presents an efficient method for accurately reconstructing array antenna signals against a noisy backdrop.

Microbial methanogenesis in aerobic water: A key driver of surface methane enrichment in a deep reservoir.

Significant amounts of the greenhouse gas methane (CH4) are released into the atmosphere worldwide via freshwater sources. The surface methane maximum (SMM), where methane is supersaturated in surface water, has been observed in aquatic systems and contributes significantly to emissions. However, little is known about the temporal and spatial variability of SMM or the mechanisms underlying its development in artificial reservoirs. Here, the community composition of methanogens as major methane producers in the water column and the mcrA gene was investigated, and the cause of surface methane supersaturation was analyzed. In accordance with the findings, elevated methane concentration of SMM in the transition zone, with an annually methane emission flux 2.47 times higher than the reservoir average on a large and deep reservoir. In the transition zone, methanogens with mcrA gene abundances ranging from 0.5 × 103-1.45 × 104 copies/L were found. Methanobacterium, Methanoseata and Methanosarcina were the three dominate methanogens, using both acetic acid and H2/CO2 pathways. In summary, this study contributes to our comprehension of CH4 fluxes and their role in the atmospheric methane budget. Moreover, it offers biological proof of methane generation, which could aid in understanding the role of microbial methanogenesis in aerobic water.

Polydopamine-based nano adjuvant as a promising vaccine carrier induces significant immune responses against Acinetobacter baumannii-associated pneumonia.

The objective of this study was to assess the effectiveness of polydopamine nanoparticles (PDANPs) as a delivery system for intranasal antigen administration to prevent Acinetobacter baumannii (A. baumannii)-associated pneumonia. In the in vitro phase, the conserved outer membrane protein 22 (Omp22)-encoding gene of A. baumannii was cloned, expressed, and purified, resulting in the production of recombinant Omp22 (rOmp22), which was verified using western blot. PDANPs were synthesized using dopamine monomers and loaded with rOmp22 through physical adsorption. The rOmp22-loaded PDANPs were characterized in terms of size, size distribution, zeta potential, field emission scanning electron microscopy (FESEM), loading capacity, Fourier transform infrared spectroscopy (FTIR), release profile, and cytotoxicity. In the in vivo phase, the adjuvant effect of rOmp22-loaded PDANPs was evaluated in terms of eliciting immune responses, including humoral and cytokine levels (IL-4, IL-17, and IFN-γ), as well as protection challenge. The rOmp22-loaded PDANPs were spherical with a size of 205 nm, a zeta potential of -14 mV, and a loading capacity of approximately 35.7 %. The released rOmp22 from nontoxic rOmp22-loaded PDANPs over 20 days was approximately 41.5 %, with preserved rOmp22 integrity. The IgG2a/IgG1 ratio and IFN-γ levels were significantly higher in immunized mice with rOmp22-loaded-PDANPs than in rOmp22-alum, naive Omp22, and control groups. Furthermore, rOmp22-loaded PDANPs induced effective protection against infection in the experimental challenge and showed more normal structures in the lung histopathology assay. The results of this study suggest the potential of PDANPs as a nano-adjuvant for inducing strong immune responses to combat A. baumannii.

Subcutaneous application of hyperimmune serum against Histophilus somni recombinant proteins affects serum antibody reactivity in beef calves.

BACKGROUND: Respiratory tract diseases cause significant economic loss in beef cattle. This study aimed to determine whether the application of hyperimmune serum (HS) containing antibodies against selected antigens of Gram-negative bacteria would improve the health and growth of different breeds of beef calves kept on three farms. Two recombinant protein antigens (Histophilus somni rHsp60 and rOMP40) were used to immunize four cows to produce HS. Eighty seven beef calves (Charolaise n = 36, Limousine n = 34, and crossbreed n = 17) were included into study. One hundred milliliters of serum were administered subcutaneously to 43 beef calves (Charolaise n = 18, Limousine n = 17, and crossbreed n = 8) twice, between 1 and 5 and 21-28 days of life. Calves were examined three times, and blood samples were taken to evaluate immunoglobulin M, G1, and G2, fibrinogen, serum amyloid A, and haptoglobin concentrations and reactivity of these Ig classes of antibodies against H. somni rHsp60 and rOMP40. Average daily weight gain during the first month and until weaning was calculated. RESULTS: HS showed higher (p ≤ 0.05) reactivity in calf sera against H. somni rHsp60 and OMP40 in IgG1 and IgG2. In experimental calves, compared to control calves, the reactivity of IgG1 against rOMP40 in the second sampling was higher in Limousine calves (p ≤ 0.001) and in the other two herds (p ≤ 0.05). Serum IgG2 antibody activity against H. somni rHsp60 in the second sampling was higher in experimental calves than in control calves in charolaise (p ≤ 0.05) and limousine (p ≤ 0.001) herds. The reactivity of IgG2 against rOMP40 in the second sampling of experimental calves was higher in herds with Charolaise and Limousine calves (p ≤ 0.001) and in crossbred calves (p ≤ 0.05). In the third sampling, serum IgG1 antibody reactivity against rOMP40 in Limousine calves was higher (p ≤ 0.05) in the experimental group. Among the other evaluated parameters, only SAA in the second sampling in the herd with Charolaise calves and heart rate in the herd with Limousine calves were significantly higher in the control calves (p ≤ 0.05). CONCLUSION: The application of HS to calves in all herds had an impact on specific reactivity in IgG1 and IgG2 classes against H. somni rOMP40 and rHsp60, antigens which were used for serum production.

Design of an oral vaccine using Lactococcus lactis against brucellosis: an in vitro and in vivo study.

Brucellosis is regarded as one of the world's most severe zoonotic diseases. This study aimed to investigate the possibility of using recombinant Lactococcus lactis (L. lactis) as a live vector to produce recombinant Brucella abortus (B. abortus) Omp10. The gene sequences were obtained from GenBank. The proteins' immunogenicity was assessed using Vaxijen. After confirming the cloning of the Omp10 gene in the pNZ8148 vector by enzymatic digestion and PCR, transformation into L. lactis was done. SDS-PAGE and western blot methods evaluated omp10 protein expression. Mice received oral recombinant L. lactis vaccines. IgG antibodies against Omp10 were tested using ELISA. Real-time PCR and ELISA were used to analyze cytokine responses. Survival rate and histopathological changes were evaluated after the challenge. Omp10 was chosen for its 1.5524 antigenicity score. Enzymatic digestion and PCR identified a 381-bp gene fragment. A 10 kDa band indicated the success of L. lactis transformation. Mice administered the L. lactis-pNZ8148-Omp10-Usp45 vaccination 14 days after priming showed significantly higher Omp10-specific total IgG and IgG1 (P < 0.001) than the PBS control group. The mice who received the L. lactis-pNZ8148-Omp10-Usp45 and IRBA vaccines had significantly elevated levels of IFN-γ, TNFα, IL-4, and IL-10 in samples collected on days 14 and 28 (P < 0.001). Inflammatory response, morphological damage, alveolar edema, and lymphocyte infiltration were reduced in the target group. A recombinant L. lactis expressing the Omp10 protein was constructed as an oral Lactococcus-based vaccine and compared to live attenuated vaccines for future brucellosis investigations.

ATP-independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next-generation therapeutics.

Diderm bacteria employ ß-barrel outer membrane proteins (OMPs) as their first line of communication with their environment. These OMPs are assembled efficiently in the asymmetric outer membrane by the ß-Barrel Assembly Machinery (BAM). The multi-subunit BAM complex comprises the transmembrane OMP BamA as its functional subunit, with associated lipoproteins (e.g., BamB/C/D/E/F, RmpM) varying across phyla and performing different regulatory roles. The ability of BAM complex to recognize and fold OM ß-barrels of diverse sizes, and reproducibly execute their membrane insertion, is independent of electrochemical energy. Recent atomic structures, which captured BAM-substrate complexes, show the assembly function of BamA can be tailored, with different substrate types exhibiting different folding mechanisms. Here, we highlight common and unique features of its interactome. We discuss how this conserved protein complex has evolved the ability to effectively achieve the directed assembly of diverse OMPs of wide-ranging sizes (8-36 ß-stranded monomers). Additionally, we discuss how darobactin-the first natural membrane protein inhibitor of Gram-negative bacteria identified in over five decades-selectively targets and specifically inhibits BamA. We conclude by deliberating how a detailed deduction of BAM complex-associated regulation of OMP biogenesis and OM remodeling will open avenues for the identification and development of effective next-generation therapeutics against Gram-negative pathogens.

Anti-NOTCH1 therapy with OMP-52 M51 inhibits salivary adenoid cystic carcinoma by depressing epithelial-mesenchymal transition (EMT) process and inducing ferroptosis.

Salivary adenoid cystic carcinoma (ACC) is a common type of salivary gland cancer, and the mechanisms underlying its progression still remain poorly understood without efficient therapies. NOTCH1, an evolutionally conserved cell-cell signaling pathway, is involved in the progression of ACC. In our study, we attempted to explore whether NOTCH1 suppression using the monoclonal anti-NOTCH1 antibody OMP-52 M51 could be of potential for ACC treatment. Here, we identified NOTCH1 elevation in human ACC tissues compared with the matched normal samples. Patients with metastasis expressed much higher NOTCH1. We then found that OMP-52 M51 markedly reduced the expression of NOTCH1 and its intracellular active form NICD1 (NOTCH1 intracellular domain). Importantly, OMP-52 M51 markedly reduced the proliferation, migration and invasion of ACC cells. RNA-Seq and in vitro studies further showed that OMP-52 M51 significantly induced ferroptosis in ACC cells, indicated by the increased cellular malondialdehyde (MDA), iron contents and lipid ROS production, and decreased glutathione (GSH) levels. Further, remarkable glutathione peroxidase 4 (GPX4) reduction was detected in ACC cells with OMP-52 M51 treatment. However, promoting NOTCH1 expression markedly abolished the function of OMP-52 M51 to induce ferroptosis. Intriguingly, low-dose OMP-52 M51 strongly facilitated the capacity of ferroptosis inducer erastin to trigger ferroptotic cell death, revealing that OMP-52 M51 could improve the sensitivity of ACC cells to ferroptosis. In vivo, OMP-52 M51 administration suppressed tumor growth and induced ferroptosis in the constructed ACC xenograft mouse model. Collectively, our findings demonstrated that NOTCH1 inhibition by OMP-52 M51 represses the proliferation and epithelial-mesenchymal transition (EMT) in ACCs, and promotes ferroptosis, revealing the potential therapeutical application of OMP-52 M51 in ACC.

Bioremediation of rapid sand filters for removal of organic micropollutants during drinking water production.

Rapid sand filtration (RSF) is used during drinking water production for removal of particles, possible harmful microorganisms, organic material and inorganic compounds such as iron, manganese, ammonium and methane. However, RSF can also be used for removal of certain organic micropollutants (OMPs). In this study, it was investigated if OMP removal in columns packed with sand from full scale RSFs could be stimulated by bioaugmentation (i.e. inoculating RSFs with sand from another RSF) and/or biostimulation (i.e. addition of nutrients, vitamins and trace-elements that stimulate microbial growth). The results showed that removal of PFOA, carbamazepine, 1-H benzotriazole, amidotrizoate and iopamidol in the columns was low (< 20 %). Propranolol and diclofenac removal was higher (50-60 %) and propranolol removal likely occurred via sorption processes, whereas for diclofenac it was unclear if removal was a combination of physical-chemical and biological processes. Moreover, bioaugmentation and biostimulation resulted in 99 % removal of gabapentin and metoprolol after 38 days and 99 % removal of acesulfame after 52 days of incubation. The bioaugmented column without biostimulation showed 99 % removal for gabapentin and metoprolol after 52 days, and for acesulfame after 80 days. In contrast, the non-bioaugmented column did not remove gabapentin, removed < 40 % metoprolol and showed 99 % removal of acesulfame only after 80 days of incubation. Removal of these OMPs was negatively correlated with ammonium oxidation and the absolute abundance of ammonia-oxidizing bacteria. 16S rRNA gene sequencing showed that OMP removal of acesulfame, gabapentin and metoprolol was positively correlated to the relative abundance of specific bacterial genera that harbor species with a heterotrophic and aerobic or denitrifying metabolism. These results show that bioaugmentation of RSF can be successful for OMP removal, where biostimulation can accelerate this removal.

Effects of Pasturella Multocida B: 2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes / Efeitos de Pasteurella multocida B: 2 e seus imunógenos (LPS e OMP) em hormônios reprodutivos em búfalos Nili-Ravi

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P<0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a "septicemia hemorrágica" (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P < 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

Effects of Pasturella Multocida B:2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P 0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a septicemia hemorrágica (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

OmpC and OmpF Outer Membrane Proteins of Escherichia coli and Salmonella enterica Form Bona Fide Amyloids.

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the ß-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their ß-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-ß that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.