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Integrated dual transcriptome sequencing and experimental validation reveal potential mechanisms of baicalin against pneumocystis pneumonia in immunosuppressed rats.

BACKGROUND: Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality in immunocompromised individuals. Although baicalin (Ba), a natural bioactive flavonoid, has demonstrated protective and therapeutic effects against PCP, its molecular mechanisms remain undefined. We employed dual RNA sequencing (dual RNA-seq) to characterize host and pathogen transcriptional responses to Ba treatment in an immunosuppressed rat model of PCP. METHODS: Comparative transcriptomic analyses identified differentially expressed genes in both the host and Pneumocystis, followed by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analyses. Candidate targets were further investigated using network pharmacology, protein-protein interaction analysis, molecular docking, and molecular dynamics simulations. Key findings were validated by immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative PCR. RESULTS: Ba markedly remodeled host and pathogen transcriptomes. Host transcriptomic analyses showed that Ba attenuated inflammatory and oxidative stress responses by modulating immune-related pathways, including Toll-like receptor, NF-κB, cytokine-cytokine receptor interaction, chemokine signaling, Th17 cell differentiation, and antigen processing and presentation. Experimental validation demonstrated that Ba reduced pulmonary expression of indoleamine 2,3-dioxygenase 1 (IDO1), Toll-like receptor 2 (TLR2), and TLR4 while increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant enzyme heme oxygenase-1 (HO-1). Pathogen transcriptomic analysis identified Pneumocystis Rtt109 (PcRtt109), a fungal histone acetyltransferase, as a potential pathogen-specific target that was significantly downregulated after Ba treatment. Molecular docking and molecular dynamics simulations supported stable interactions between Ba and IDO1, Nrf2, TLR2, TLR4, and PcRtt109, with the strongest predicted binding observed for PcRtt109. CONCLUSION: Dual RNA-seq revealed that Ba exerts anti-PCP activity through coordinated modulation of host and pathogen molecular networks. Its therapeutic effects are associated with suppression of inflammatory signaling, enhancement of antioxidant defenses, and inhibition of a fungal virulence-associated target. These findings provide mechanistic insights into host-pathogen interactions during PCP and support Ba as a potential therapeutic candidate for PCP.

Nrf2↗

The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation.

Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly. The C terminus of nebulin, located in the sarcomere Z-disk, comprises an SH3 domain, a module well known for its role in protein/protein interactions. SH3 domains are known to recognize proline-rich ligands, which have been classified as type I or type II, depending on their relative orientation with respect to the SH3 domain in the complex formed. Type I ligands are bound with their N terminus at the RT loop of the SH3 domain, while type II ligands are bound with their C terminus at the RT loop. Many SH3 domains can bind peptides of either class. Despite the potential importance of the SH3 domain for the function of nebulin as an integral part of a complex network of interactions, no in vivo partner has been identified so far. We have adopted an integrated approach, which combines bioinformatic tools with experimental validation to identify possible partners of nebulin SH3. Using the program SPOT, we performed an exhaustive screening of the muscle sequence databases. This search identified a number of potential nebulin SH3 partners, which were then tested experimentally for their binding affinity. Synthetic peptides were studied by both fluorescence and NMR spectroscopy. Our results show that nebulin SH3 domain binds selectively to type II peptides. The affinity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 microM). This affinity is among the highest found for SH3/peptide complexes, suggesting that the identified stretch could have significance in vivo. The strategy outlined here is of more general applicability and may provide a valuable tool to identify potential partners of SH3 domains and of other peptide-binding modules.

Amino Acid Sequence↗

Multi-omics and experimental validation identify RAPGEF2 as a protective prognostic biomarker in clear cell renal cell carcinoma.

Kidney Renal Clear Cell Carcinoma (KIRC) is characterized by marked molecular heterogeneity and metabolic reprogramming, underscoring the need for reliable biomarkers for prognostic assessment and individualized treatment. RAPGEF2, a guanine nucleotide exchange factor has been implicated in cell adhesion and differentiation, but its role in KIRC remains unclear. In this study, we systematically evaluated the expression pattern, prognostic significance, genomic associations, biological function, and therapeutic relevance of RAPGEF2 in KIRC through integrated multi-omics analyses and experimental validation. Pan-cancer single-cell and Spatial transcriptomic analysis revealed heterogeneous RAPGEF2 expression across tumor types, with a relatively prominent signal in KIRC, where RAPGEF2 was mainly enriched in endothelial cells. Survival analyses in the TCGA-KIRC showed that high RAPGEF2 expression was significantly associated with favorable overall survival, disease-specific survival, and progression-free interval, and these findings were validated in independent ICGC_RECA-EU and E-MTAB-1980 cohorts. Multivariate Cox regression further confirmed RAPGEF2 as an independent protective prognostic factor. Immunohistochemistry in a tissue microarray cohort demonstrated that higher RAPGEF2 protein expression was associated with improved overall survival. Genomic analyses showed that low RAPGEF2 expression was related to higher mutational burden. Functional assays demonstrated that RAPGEF2 knockdown promoted KIRC progression. Enrichment analyses indicated that RAPGEF2 may be associated with metabolic pathway remodeling, while immunotherapy cohort analyses suggested its potential association with therapeutic benefit. Collectively, RAPGEF2 is identified as a protective prognostic biomarker and potential functional regulator in KIRC.

Biomarker↗

Experimental validation of the use of Kramers-Kronig relations to eliminate the phase sheet ambiguity in broadband phase spectroscopy.

The technique of broadband phase spectroscopy proposed in 1978 by Sachse and Pao [J. Appl. Phys. 49, 4320-4327 (1978)] determines the phase velocity as a function of frequency from the Fourier transforms of a received reference and through-sample signal. Although quite successful, this approach can be influenced by an ambiguity in the phase velocity calculation which stems from the boundedness of the inverse tangent operation used to calculate phase. Several empirical approaches to resolve the phase ambiguity have been reported. An alternative approach that has not previously been considered appeals to the causal nature of the measurements. This article experimentally validates a method which uses the causally consistent Kramers-Kronig relations to eliminate the ambiguity in phase spectroscopy-derived phase velocity calculations. Broadband pulse and narrow-band tone burst measurements were performed on three gelatin-based phantoms containing different concentrations of graphite particles (0%, 10%, and 20% by volume). The phantoms were constructed to have attenuation coefficients which vary approximately linear-with-frequency, a dependence exhibited by many soft tissues. The narrow-band phase velocity measurements do not suffer from a phase ambiguity, and thus they serve as a "gold standard" against which the broadband phase velocity measurements are compared. The experimental results illustrate that using the Kramers-Kronig dispersion relations in conjunction with phase spectroscopy-derived phase velocity measurements is an effective means by which to resolve the phase sheet ambiguity in broadband phase spectroscopy.

Fourier Analysis↗

Toward quantitative simulation of germinal center dynamics: biological and modeling insights from experimental validation.

As models of immune system dynamics are developed, it is important to validate them with specific experimental data in order to understand their shortcomings and guide them toward becoming predictive. In this paper, we examine whether a particular mathematical model of germinal center dynamics, proposed by Oprea and Perelson, can reproduce experimental data from two specific primary responses, namely those directed against the haptens 2-phenyl-5-oxazolone and (4-hydroxy-3-nitrophenyl)acetyl. We develop formulas for estimating response-specific model parameters, as well as constraints for validating the model. In addition, we outline a general methodology for translating a continuous/deterministic model, expressed as a set of ordinary differential equations, into a discrete/stochastic framework. This methodology is used to create a new implementation of the Oprea and Perelson model that enables comparison with data on individual germinal centers. We conclude that while the model can reproduce the average dynamics of splenic germinal centers, it is at best incomplete and does not reproduce the distribution of individual germinal center behaviors. In addition to suggesting possible extensions to the model which can reconcile the dynamics with some aspects of the experimental data, we make a number of specific predictions that can be tested by in vivo experiments to obtain further insights and validation.

Antibody Affinity↗

A nonlinear finite element model of the eye with experimental validation for the prediction of globe rupture.

Over 2.4 million eye injuries occur each year in the US, with over 30,000 patients left blind as a result of the trauma. The majority of these injuries occur in automobile crashes, military operations and sporting activities. This paper presents a nonlinear finite element model of the eye and the results of 22 experiments using human eyes to validate for globe rupture injury prediction. The model of the human eye consists of the cornea, sclera, lens, ciliary body, zonules, aqueous humor and vitreous body. Lagrangian membrane elements are used for the cornea and sclera, Lagrangian bricks for the lens, ciliary, and zonules, and Eulerian brick elements comprise the aqueous and vitreous. Nonlinear, isotropic material properties of the sclera and cornea were gathered from uniaxial tensile strip tests performed up to rupture. Dynamic modeling was performed using LS-Dyna. Experimental validation tests consisted of 22 tests using three scenarios: impacts from foam particles, BB's, and baseballs onto fresh eyes used within 24 hours postmortem. The energies of the projectiles were chosen so as to provide both globe rupture and no rupture tests. Displacements of the eye were recorded using high speed color video at 7100 frames per second. The matched simulations predicted rupture of the eye when rupture was seen in the BB and baseball tests, and closely predicted displacements of the eye for the foam tests. Globe rupture has previously been shown to occur at peak stresses of 9.4 MPa using the material properties included in the model. Because of dynamic effects and improvements in boundary conditions resulting from a more realistic modeling of the fluid in the anterior and posterior chambers, the stresses can be much higher than those previously predicted, with the globe remaining intact. The model is empirically verified to predict globe rupture for stresses in the corneoscleral shell exceeding 23 MPa, and local dynamic pressures exceeding 2.1 MPa. The model can be used as a predictive aid to reduce the burden of eye injury, and can serve as a validated model to predict globe rupture.

Journal Article↗

Endosonographic Doppler-guided manometry of esophageal varices: experimental validation and clinical feasibility.

BACKGROUND AND STUDY AIMS: The risk of variceal bleeding cannot be accurately predicted using endoscopy alone. Although variceal pressure has been demonstrated to be a major determinant for the rupture of esophageal varices, direct determination by needle puncture is unsuitable for routine clinical use. Due to their operator-dependency, current noninvasive endoscopic methods for determination of variceal pressure have not gained wide acceptance. We have developed a new method of measuring variceal pressure, using endoscopic power Doppler imaging to monitor the manometry of esophageal varices. The aims of this study were to test in vitro the accuracy of Doppler-guided manometry and to assess the clinical feasibility of this method. MATERIALS AND METHODS: Experimental validation of this technique was performed using an in vitro model of artificial varices of different sizes. A linear-array endosonography (EUS) probe with power Doppler capability was used to assess flow in the varices and a balloon for manometry of esophageal varices was attached to the tip of the probe. Pressure readings were made at the time of disappearance of the Doppler signal during variceal compression by the balloon. Linear regression analysis was used to compare the results of Doppler-guided and direct intraluminal pressure measurement in the artificial varices. Variceal pressure was then measured with this technique in 28 patients with portal hypertension and esophageal varices without previous bleeding, and the results were compared with portal pressure assessed according to the hepatic vein pressure gradient (HVPG). RESULTS: In vitro studies demonstrated a good correlation between the pressure measured with Doppler monitoring and the actual intravariceal pressure (r > or = 0.922; P < 0.001). The determination of variceal pressure with this method was technically successful in 26/28 patients (93 %). The intraoperator variance was 9.3 +/- 8.6 %. Overall, the mean variceal pressure was significantly lower than the mean HVPG (21.2 +/- 5.3 mmHg vs. 24.3 +/- 7.8 mmHg; P < 0.01). Variceal pressure and portal pressure (as assessed by the HVPG) correlated significantly (r = 0.64; P < 0.001). CONCLUSIONS: Our preliminary results indicate that EUS Doppler-guided manometry of esophageal varices is feasible and accurate. This technique may become a more reliable method for noninvasive measurement of variceal pressure and warrants further investigation.

Endosonography↗

HNRNPC as a Novel Therapeutic Target for Ischemic Heart Disease: Evidence From Mendelian Randomization and Experimental Validation.

BACKGROUND: Several studies have suggested that N6-methyladenosine (m6A) plays an essential role in cardiovascular disease, but the causality of m6A on ischemic heart disease (IHD) remains unknown. Therefore, this study investigated the potential relationship between m6A and IHD using a 2-sample Mendelian randomization method. METHODS: The publicly available genome-wide association study data for m6A-related proteins were obtained from the INTERVAL study, a large population-based cohort of healthy blood donors in the United Kingdom, whereas the genome-wide association study database (including 30&#x2009;952 cases and 187&#x2009;840 healthy controls) provided the IHD data. We performed a 2-sample Mendelian randomization analysis to evaluate the potential causal association between HNRNPC (heterogeneous nuclear ribonucleoprotein C) and IHD, followed by experimental validation in&#xa0;vitro and in&#xa0;vivo to confirm the role of HNRNPC in IHD pathogenesis. RESULTS: There was no indication of pleiotropy or heterogeneity among the 6 m6A-associated proteins, but Mendelian randomization analysis revealed that HNRNPC (odds ratio [OR], 0.93 [95% CI, 0.88-0.97]; P=0.002) was associated with IHD. When IHD developed, there was a significant upregulation of HNRNPC expression in both animal and cellular tests. HNRNPC knockdown prevented oxidative stress, mitochondrial dysfunction, and cell death. CONCLUSIONS: The Mendelian randomization study suggests a potential causal association of the m6A-related protein HNRNPC in the cause of IHD and verified the accuracy of the results through a series of experiments, which will help us understand the pathogenesis of IHD and identify potential therapeutic targets in the future.

Humans↗

Experimental validation of two elastodynamic models for the wave field generated by ultrasonic transducers.

Two different three-dimensional elastodynamic models are introduced to simulate the wave field generated in steel by two types of surface mounted ultrasonic transducers. By replacing the actual transducer by an equivalent surface source distribution, the models become amenable to an exact analytical analysis. The first model simulates the action of a contact transducer through a distribution of nonmoving line segment sources. The second model simulates the action of an angle beam transducer through a single moving line segment source. Almost any transducer aperture shape may be modeled, while the source may apply a nonuniform traction. To speed up the numerical space-time domain calculations, the Cagniard-De Hoop method is employed to analytically evaluate the wave field produced by a single nonmoving line segment source. This solution provides the integrand for both single-integral models. The models are experimentally validated for a contact transducer and three different angle beam transducers. The validation involves a comparison of the wave-field patterns, the directivity curves and some time-domain signals from the wave field. It is shown that the models reliably identify the wide variety of waves generated by ultrasonic transducers, such as focused waves, edge waves, Rayleigh waves and head waves.

Journal Article↗

Non-professional paint stripping, model prediction and experimental validation of indoor dichloromethane levels.

We have experimentally quantified exposure to dichloromethane during non-professional paint stripping and validated the mathematical paint exposure model of van Veen et al. (1999). The model innovates the prediction of the dichloromethane evaporation rate and room concentration by accounting for transport in the paint stripper matrix. The experiments show that peak concentrations range from 600 to 1600 mg/m3, increasing to 2000 mg/m3 when direct sun radiation increases evaporation. A naive model prediction, using a priori parameter values from the experimental set-up and a previous experiment with alkanes, accurately predicts the upper range of the experimental values, but overpredicted four out of six experiments. Statistical fit of the two paint stripper layer parameters to the experimental data resulted in a good coincidence of predicted and experimental data. Model and experiment indicate that 10-30% of dichloromethane is immediately available for evaporation.

Air Pollution, Indoor↗

Pattern dynamics of vortex ripples in sand: nonlinear modeling and experimental validation.

Vortex ripples in sand are studied experimentally in a one-dimensional setup with periodic boundary conditions. The nonlinear evolution, far from the onset of instability, is analyzed in the framework of a simple model developed for homogeneous patterns. The interaction function describing the mass transport between neighboring ripples is extracted from experimental runs using a recently proposed method for data analysis, and the predictions of the model are compared to the experiment. An analytic explanation of the wavelength selection mechanism in the model is provided, and the width of the stable band of ripples is measured.

Journal Article↗

Experimentally validated 3-D simulation of shock waves generated by dense explosives in confined complex geometries.

Accidental blast wave generation and propagation in the surroundings poses severe threats for people and property. The prediction of overpressure maxima and its change with time at specified distances can lead to useful conclusions in quantitative risk analysis applications. In this paper, the use of a computational fluid dynamics (CFD) code CFX-5.6 on dense explosive detonation events is described. The work deals with the three-dimensional simulation of overpressure wave propagation generated by the detonation of a dense explosive within a small-scale branched tunnel. It also aids at validating the code against published experimental data as well as to study the way that the resulting shock wave propagates in a confined space configuration. Predicted overpressure histories were plotted and compared versus experimental measurements showing a reasonably good agreement. Overpressure maxima and corresponding times were found close to the measured ones confirming that CFDs may constitute a useful tool in explosion hazard assessment procedures. Moreover, it was found that blast wave propagates preserving supersonic speed along the tunnel accompanied by high overpressure levels, and indicating that space confinement favors the formation and maintenance of a shock rather than a weak pressure wave.

Chemical Industry↗

Mechanism of Qigu capsule as a treatment for sarcopenia based on network pharmacology and experimental validation.

OBJECTIVE: To explore the potential molecular mechanism of Qigu capsule (&#xff0c;QGC) in the treatment of sarcopenia through network pharmacology and to verify it experimentally. METHODS: The active compounds of QGC and common targets between QGC and sarcopenia were screened from databases. Then the herbs-compounds-targets network, and protein-protein interaction (PPI) network was constructed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by R software. Next, we used a dexamethasone-induced sarcopenia mouse model to evaluate the anti-sarcopenic mechanism of QGC. RESULTS: A total of 57 common targets of QGC and sarcopenia were obtained. Based on the enrichment analysis of GO and KEGG, we took the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway as a key target to explore the mechanism of QGC on sarcopenia. Animal experiments showed that QGC could increase muscle strength and inhibit muscle fiber atrophy. In the model group, the expression of muscle ring finger-1 and Atrogin-1 were increased, while myosin heavy chain was decreased, QGC treatment reversed these changes. Moreover, compared with the model group, the expressions of p-PI3K, p-Akt, p-mammalian target of rapamycin and p-Forkhead box O3 in the QGC group were all upregulated. CONCLUSION: QGC exerts an anti-sarcopenic effect by activating PI3K/Akt signaling pathway to regulate skeletal muscle protein metabolism.

Sarcopenia↗

Measurement of CBF and CMRO2 using the continuous inhalation of C15O2 and 15O. Experimental validation using CO2 reactivity in the anaesthetised dog.

Experimental support for the steady state 15O inhalation technique, as used to measure cerebral blood flow (CBF) and oxygen utilisation (CMRO2), was obtained by describing the response of the cerebral vasculature to variations in arterial PCO2 in 6 anaesthetised dogs. Measurements were made using a positron emission tomography (ECAT II) and arterial blood sampling, during the sequential constant inhalation of C15O2 and 15O2. Values of CBF and CMRO2 were calculated for a mixture of white and grey matter, using the steady state tracer equations derived by Jones et al. (1976). The mean CMRO2 was 3.58 +/- 0.81 ml O2 . 100 ml-1 . min-1, whilst the mean CBF and OER (oxygen extraction ratio) values (for an arterial PCO2 of 40 mm Hg) were 39.9 ml . 100 ml-1 and 0.50 +/- 0.06, respectively. Arterial PCO2 was varied between 20 and 150 mm Hg. CBF was found to correlate closely with arterial PCO2, resulting in a mean slope (specific reactivity) of 1.52 +/- 0.38 ml . 10 ml-1. mm Hg-1. Pooling the flow data resulted in a linear relationship between CBF (% change) and arterial PCO2 in the range 20-70 mm Hg, with a slope (% reactivity) of 3.2% mm Hg-1 (2 P less than 0.001). The oxygen extraction ratio (OER) fell with increasing values of arterial PCO2 resulting in a stable CMRO2 throughout each study. There was no correlation between CMRO2 and artificially increased CBF. These results support and give confidence in the use of the 15O inhalation technique for measuring CBF, OER and CMRO2.

Animals↗

Experimental validation of a new ultrasound method for the simultaneous assessment of radial and longitudinal myocardial deformation independent of insonation angle.

BACKGROUND: Strain and strain rate have been proposed as tools to quantify regional myocardial function. One of the major pitfalls of the current methodology is its angle dependency. To overcome this problem, we have developed a new method for the estimation of strain, independent of angle. The aim of this study was to validate this new methodology in an experimental setting using sonomicrometry. METHODS AND RESULTS: In 5 open-chest sheep, ultrasound data were acquired. The new methodology was used to perform simultaneous measurements of radial and longitudinal strain in the inferolateral wall. Segment-length sonomicrometry crystals were used as the reference. After baseline acquisitions, deformation was modulated by pharmacologically changing the inotropic state of the myocardium and by inducing ischemia. Ultrasonically estimated radial and longitudinal strain were validated against sonomicrometry by means of Bland-Altman analysis and the intraclass correlation coefficient. For both strain components, good agreements were found between the ultrasound and the sonomicrometry measurements as shown by Bland-Altman statistics. The intraclass correlation coefficients were found to be 0.72 and 0.80 for the radial and longitudinal components, respectively. CONCLUSIONS: A new technique for the estimation of myocardial deformation was validated. It was shown that the current problem of angle dependency was solved and that 2 deformation components could be estimated simultaneously and accurately. Furthermore, the technique was less time-consuming, because anatomic tracking was performed automatically. This approach could potentially accelerate the clinical acceptance of ultrasound deformation imaging in cardiology.

Animals↗

Analytical calculation of the portal scatter to primary dose ratio: an EGS4 Monte Carlo and experimental validation at large air gaps.

An analytical approximation for the scatter to primary dose ratio (SPR) on the central axis was validated against Monte Carlo results and experimental measurements for homogeneous and inhomogeneous phantoms. The analytical approximation only included first-order Compton scatter. The contribution to the total SPR from first-order Compton scatter, multiply scattered photons and electron scatter was investigated using Monte Carlo simulation for homogeneous phantoms (up to 30 cm thick for 6 and 18 MV beams; source to detector distances from 150 to 230 cm) as well as for a neck, thorax and pelvis phantom. SPRs were measured on the central axis with an ionization chamber for water phantoms (up to 20 cm thick at 4 MV, 30 cm for 6 MV and 10 MV and 40 cm for 18 MV; source to detector distances of 185 and 200 cm) and for phantoms representing the neck, thorax and pelvis (for air gaps of 50 cm and larger). The mean difference between the experimental and analytical SPRs on the central axis for source to detector distances of 170 cm or greater was within: -0.003 (neck); -0.012 (thorax); -0.028 (pelvis, 10 MV) and 0.008 (pelvis, 18 MV) respectively.

Computer Simulation↗

[Experimental validation of the method of autografts creation for arteries].

Experimental studies on rats have demonstrated possibility of connective-tissue grafts formation on polyvinylchloride base introduced subcutaneously. Dynamics of autograft formation is characterized by gradual thinning of wall, regulation of collagen fibers, decreased number of cells with fibrocytes predominance. Autografts in carotid position retain its patency during 3 months, and endotheliocytes appear on their internal surface.

Animals↗

[The experimental validation of associated immunization with paired combinations of vaccines].

Experimental researches were conducted on reactogenicity and immunological efficiency of pair associations of 8 commercial drug preparations. The following vaccines were used in various pair combinations: typhoid, plague, smallpox, typhus, tick-borne encephalitis, yellow-fever, cholera, sextaanatoxine. Pair combinations of these drug preparations were proved to be harmless. Their reactogenicity was manifested mainly in more or less pronounced vaccination reaction which may be considered as tolerable. Immunological efficiency practically of all pair combinations of vaccines, which was determined by the frequency and intensity of antibody formation, had the same indexes as the control one. The immunity expression of a large majority of pair combinations of vaccines also was unchanged. The study in vitro of compatibility of pair associations of vaccines proves the low stability of yellow-fever vaccine, which falls down under the influence of plague and cholera vaccines, or sextaanatoxine. On the contrary, smallpox vaccine makes yellow-fever vaccine more active. Plague vaccine is stimulated by smallpox vaccine and is oppressed with combined application of typhoid vaccine and sextaanatoxine.

Animals↗