Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Septicemic salmonellosis in suckling piglets resulting from improper intramuscular administration of an oral vaccine.

We describe an unusual outbreak of mortality in suckling piglets following the misadministration of an oral vaccine against Salmonella Typhimurium and Salmonella Choleraesuis. Within 3-48 h of vaccination of a batch of ~700 piglets, ~300 developed marked swelling in the dorsal neck region, respiratory distress, fever, recumbency, and apathy. In total, ~100 died, and 4 were submitted for autopsy. Gross and microscopic lesions consisted of focally extensive areas of purple discoloration in the skin of the cervical region, associated with edema and hemorrhage in the subcutis and muscles. Additionally, there was interstitial pneumonia with marked interlobular edema and mild fibrinous pleuritis. Aerobic bacterial culture identified Salmonella Typhimurium (3 cases) and Salmonella Choleraesuis (1 case) in samples of skeletal muscle and lung and from pleural swab samples. Marked immunostaining against Salmonella spp. was observed in the skeletal muscle of the cervical region, as well as in blood vessels and macrophages from the lung, liver, spleen, and kidney. We concluded that inappropriate intramuscular administration of an oral vaccine against Salmonella resulted in septicemia and death in a batch of piglets.

BrainPepPass: A Framework Based on Supervised Dimensionality Reduction for Predicting Blood-Brain Barrier-Penetrating Peptides.

Peptides that pass through the blood-brain barrier (BBB) not only are implicated in brain-related pathologies but also are promising therapeutic tools for treating brain diseases, e.g., as shuttles carrying active medicines across the BBB. Computational prediction of BBB-penetrating peptides (B3PPs) has emerged as an interesting approach because of its ability to screen large peptide libraries in a cost-effective manner. In this study, we present BrainPepPass, a machine learning (ML) framework that utilizes supervised manifold dimensionality reduction and extreme gradient boosting (XGB) algorithms to predict natural and chemically modified B3PPs. The results indicate that the proposed tool outperforms other classifiers, with average accuracies exceeding 94% and 98% in 10-fold cross-validation and leave-one-out cross-validation (LOOCV), respectively. In addition, accuracy values ranging from 45% to 97.05% were achieved in the independent tests. The BrainPepPass tool is available in a public repository for academic use (https://github.com/ewerton-cristhian/BrainPepPass).

Auricular and laryngeal chondritis in nursery and finishing pigs.

This work aimed to characterize the clinic-pathological presentation of an outbreak of auricular and laryngeal chondritis in pigs. Visits were made to pig farms, where the clinical history was obtained, and clinical and postmortem examinations were performed. In those farms, 3% to 4% of pigs presented otohematomas, which started in the nursery and extended to the finishing phase. Moreover, some finishing pigs presented with respiratory distress, initially characterized as inspiratory dyspnea, associated by an uncommon respiratory stridor and culminating in death. Grossly, nursery piglets had enlarged ears, and on the cut surface, the cartilage was fragmented and associated with blood clots. In the finishing phase, in addition to auricular lesions, the epiglottis and arytenoid cartilages were thickened and distorted, which partially occluded the lumen. Microscopically, the laryngeal and auricular cartilages were fragmented, displayed a loss of matrix basophilia, and were surrounded by lymphohistiocytic inflammatory infiltrate, with occasional multinucleated giant cells and fibrosis. The lesions exclusively affected elastic cartilages. The disease in finishing pigs led to increased mortality and was a differential diagnosis to respiratory challenges. It was not possible to determine the factor that triggered this condition; however, a nutritional association is suspected. To the authors' knowledge, this is the first report of primary auricular and laryngeal chondritis in pigs.

Temporal Structures in Positron Spectra and Charge-Sign Effects in Galactic Cosmic Rays.

We present the precision measurements of 11 years of daily cosmic positron fluxes in the rigidity range from 1.00 to 41.9 GV based on 3.4×10^{6} positrons collected with the Alpha Magnetic Spectrometer (AMS) aboard the International Space Station. The positron fluxes show distinctly different time variations from the electron fluxes at short and long timescales. A hysteresis between the electron fluxes and the positron fluxes is observed with a significance greater than 5σ at rigidities below 8.5 GV. On the contrary, the positron fluxes and the proton fluxes show similar time variation. Remarkably, we found that positron fluxes are modulated more than proton fluxes with a significance greater than 5σ for rigidities below 7 GV. These continuous daily positron fluxes, together with AMS daily electron, proton, and helium fluxes over an 11-year solar cycle, provide unique input to the understanding of both the charge-sign and mass dependencies of cosmic rays in the heliosphere.

Anatomopathological characterization of branchial cysts in slaughtered pigs.

Branchial cysts are a congenital anomaly in humans and other animal species. In this study, twenty commercially bred slaughtered pigs ranging from 120 to 150 days of age, sourced from different farms and lots, were found to have cysts in the oropharyngeal region at meat inspection despite the absence of clinical signs. Two cysts were selected for histopathological examination. The first cyst was surrounded by fibrous connective tissue and lined by a simple single cell layer of epithelium. The second cyst comprised a squamous pseudostratified to simple stratified epithelium, accompanied by a mild inflammatory infiltrate. This cyst was also surrounded by fibrous connective tissue and glands. The pathological diagnosis of branchial cysts in slaughtered pigs was established on the basis of their anatomical location and gross and microscopic findings.

Molecular characterization and in silico evaluation of surfactins produced by endophytic bacteria from Phanera splendens.

The Phanera splendens (Kunth) Vaz. is a medicinal plant that is used in traditional medicine for the treatment of various diseases, such as malaria. This plant presents highly efficient endophytic bacterial isolates with biocontrol properties. Bacillus sp. is responsible for the production of a variety of non-ribosomal synthesized cyclic lipopeptides which highlight the surfactins. Surfactins have a wide range of antimicrobial activity, including antiplasmodial activity. There is scientific evidence that surfactin structure 2d-01 can be a potent inhibitor against a Plasmodium falciparum sirtuin (Sir2) by acting on the Sir2A protein as the target. The Pf genome encodes two known sirtuins, PfSir2A and PfSir2B, where PfSir2A is a regulator of asexual growth and var gene expression. Herein, we have identified six surfactins produced by endophytic bacteria and performed in silico analysis to elucidate the binding mode of surfactins at the active site of the PfSir2A enzyme. Among the characterized surfactins, 1d-02 showed the highest affinity for the PfSir2A enzyme, with binding energy values equal to -45.08 ± 6.0 and -11.95 ± 0.8 kcal/mol, using MM/GBSA and SIE methods, respectively. We hope that the information about the surfactin structures obtained in this work, as well as the potential binding affinity with an important enzyme from P. falciparum, could contribute to the design of new compounds with antimalarial activity.

Fatal Simplexvirus humanalpha1 infection in howler-monkeys (Alouatta sp.) under human care: Clinical, molecular, and pathological findings.

BACKGROUND: Simplexvirus humanalpha1 (HuAHV-1) are common anthropozoonosis reported in marmosets but rare in howler monkeys (Alouatta sp.). METHODS: Necropsy of two brown-howler monkeys (A. caraya) and one red-howler monkey (A. guariba clamitans) from different zoo collections were performed. Fragments of all organs were examined through microscopy. Samples were submitted to IHC for Simplexvirus humanalpha 2 (HuAHV-2) [sin. Herpesvirus simplex type 2] and PCR. RESULTS: Grossly, only the A. guariba showed liver lesions characterized by multifocal, pinpoint white areas corresponding microscopically as random necrotizing herpetic hepatitis and ulcerative glossitis. Both A. caraya showed necrotizing meningoencephalitis with Cowdry A-type body inclusions within neurons and astrocytes. Immunolabeling for HuAHV-1/2 was observed in the tongue, liver, and brain. HuAHV-1 was confirmed in all samples by PCR, Sanger sequencing, and phylogenetic analyses. CONCLUSION: Necrotizing meningoencephalitis was appreciated in 2/3 of animals, and it is associated with neurologic signs. Along with ulcerative glossitis, a hallmark lesion in marmosets, it was present in one animal. Regarding herpetic hepatitis, it is not frequent in monkeys and occurs mainly in immunocompromised animals. HuAHV-1 infection was confirmed corroborating with a human source. This is the second report on captive black-howler monkeys and the first gross, histologic, immunohistochemical, and molecular description of herpetic hepatitis and ulcerative glossitis in red-howler monkeys (A. guariba).

Blocking Autophagy by the Two-Pore Channels Antagonist Tetrandrine Improves Sorafenib-Induced Death of Hepatocellular Carcinoma Cells.

Sorafenib, an oral multi-kinase inhibitor, used to treat hepatocellular carcinoma (HCC). However, drug resistance is still common in several HCC patients. This complex mechanism is not yet fully elucidated, driving the search for new therapeutic targets to potentiate the antitumoral effect of sorafenib. Recent findings have linked the expression of Two-Pore Channels (TPCs) receptors with the development and progression of cancer. TPCs receptors are stimulated by NAADP, a Ca2+ messenger, and inhibited by their antagonists Ned-19 and tetrandrine. Here, we investigate the participation of TPCs inhibition in cell death and autophagy in sorafenib-treated HCC cells. Here, we show that the association of sorafenib with tetrandrine increased sorafenib-induced cell death accompanied by increased lysotracker fluorescence intensity. In contrast, these effects were not observed after treating these cells with Ned-19. The pharmacological TPC antagonists by Ned-19 and tetrandrine or siRNA-mediated TPC1/2 inhibition decreased sorafenib-induced Ca2+ release, reinforcing the participation of TPCs in sorafenib HCC responses. Furthermore, the association tetrandrine and sorafenib blocked autophagy through ERK1/2 pathway inhibition, which represents a putative target for potentiating HCC cell death. Therefore, our study proposes the use of tetrandrine analogs with the aim of improving sorafenib therapy. Also, our data also allow us to suggest that TPCs may be a new target in anticancer therapies.

T2-fluid attenuated inversion recovery mismatch in tumefactive multiple sclerosis.

The T2-fluid attenuated inversion recovery (FLAIR) mismatch sign has been suggested as an imaging marker of isocitrate dehydrogenase-mutant 1p/19q non-codeleted gliomas with 100% specificity. Tumefactive demyelination is a common mimic of neoplasm that has led to unnecessary biopsies and even resections. We report a case of tumefactive multiple sclerosis in a 46-year-old male without prior symptomatic demyelinating episodes that demonstrates the T2-FLAIR mismatch sign. Our findings suggest the T2-FLAIR mismatch sign should not be used as a differential feature between glioma and tumefactive demyelination. Because typical isocitrate dehydrogenase-mutant 1p/19q non-codeleted gliomas typically do not demonstrate significant enhancement, such diagnosis should be reserved when post-contrast images are unavailable.

Cannabidiol Recovers Dopaminergic Neuronal Damage Induced by Reserpine or α-synuclein in Caenorhabditis elegans.

Progressive neurodegenerative disorders such as Parkinson Disease (PD) lack curative or long-term treatments. At the same time, the increase of the worldwide elderly population and, consequently, the extension in the prevalence of age-related diseases have promoted research interest in neurodegenerative disorders. Caenorhabditis elegans is a free-living nematode widely used as an animal model in studies of human diseases. Here we evaluated cannabidiol (CBD) as a possible neuroprotective compound in PD using the C. elegans models exposed to reserpine. Our results demonstrated that CBD reversed the reserpine-induced locomotor alterations and this response was independent of the NPR-19 receptors, an orthologous receptor for central cannabinoid receptor type 1. Morphological alterations of cephalic sensilla (CEP) dopaminergic neurons indicated that CBD also protects neurons from reserpine-induced degeneration. That is, CBD attenuates the reserpine-induced increase of worms with shrunken soma and dendrites loss, increasing the number of worms with intact CEP neurons. Finally, we found that CBD also reduced ROS formation and α-syn protein accumulation in mutant worms. Our findings collectively provide new evidence that CBD acts as neuroprotector in dopaminergic neurons, reducing neurotoxicity and α-syn accumulation highlighting its potential in the treatment of PD.

Memory and anxiety-like behavior of rats in the plus-maze discriminative avoidance task: Role of serotonergic transmission in the basolateral amygdala.

Optimal levels of anxiety are critical to memory consolidation, but maladaptive anxiety can disrupt memory acquisition. Serotonergic activity within the amygdala influences both anxiety-like behavior and aversive memory consolidation. To evaluate the effects of serotoninergic manipulations within the basolateral amygdala (BLA) on anxiety-like behavior and aversive memory in rats tested in the plus-maze discriminative avoidance task (PMDAT). The PMDAT investigates aversive memory and anxiety-like behavior simultaneously in rodents. Three-month-old male Wistar rats received bilateral infusions (1 µL per side) of saline, 8-OH-DPAT (5-HT1 agonist; 10 nmol), WAY100135 (5-HT1 antagonist; 0.9 nmol), ketanserine (5-HT 2 antagonist; 10 nmol), or fluoxetine (serotonin reuptake inhibitor; 1.6 nmol) into the BLA and were submitted to PMDAT training session 15 min later. In the test, 24 hr later, animals were re-exposed to the apparatus without the infusion of drugs, and aversive memory was evaluated. (a) 8-OH-DPAT did not affect memory or anxiety, but impaired avoidance behavior toward the aversive arm during training; (b) fluoxetine, WAY100135 and ketanserin impaired memory formation; (c) ketanserin decreased anxiety-like behavior; and (d) none of the treatments induced motor changes. The results showed that an increase in serotonin (5-HT) availability or the blockade of 5HT1A and 5HT2A BLA receptors impaired aversive memory formation. However, only 5HT2A receptor antagonism induced anxiolytic effects. Thus, both memory and anxiety-like behavior can be modified by changes in serotonergic transmission in the basolateral amygdala, but the effects on both phenomena seem to be mediated by different mechanisms related to serotonergic transmission. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Variations of accessory thoracic muscles identified in the ethnically diverse whole-body donation population in Northern California.

Accessory thoracic muscles in humans are relatively common and it is important to draw awareness to their variable presentations and potential clinical implications owing to their close association with the axilla. Here we report four cases of accessory thoracic muscle variations identified in the ethnically diverse whole- -body donation population in Northern California (4 out of 48 donors, 8.3%). Of these, combined presentations of thoracic accessory muscles were observed in two of the donors, one involving bilateral axillary arches and a pectoralis quartus on the left and the other a unilateral axillary arch on the left and bilateral pairs of pectoral fascicles. In the former, the proximal ends of the left axillary arch and pectoralis quartus joined to form a common aponeurosis which inserted onto the deep tendon of the pectoralis major; in the latter, the pectoral fascicles originated from the surface of the ribs and inserted into the deep surface of the pectoralis major muscle. In the other two donors, unilateral axillary arches were observed. Our observations illustrate that accessory thoracic muscles, in isolated as well as combined forms, are commonplace in the general population. We also describe the proposed embryonic origins of these accessory muscles, which may reflect their frequent occurrence, and potential clinical implications of these muscles, as discussed in literature.

Shared Decision-Making in Cardiovascular Practice.

Clinical decisions are optimally made collaboratively, with patients and clinicians working together to review all available information and treatment options. A comprehensive dialogue that identifies and brings into focus individual patient goals within the context of the evidence base is the ideal approach. Shared decision-making (SDM) is essential to making choices about treatment preferences and characterizes the optimal practice of evidence-based medicine and good patient care. By supporting patient autonomy and engagement, the patient and family become partners in their health care. Decisions surrounding whether or not to proceed with diagnostic and therapeutic procedures after fully discussing appropriate alternatives are best made considering both the evidence base and patient goals. The central feature of SDM is that a clinician and a patient engage in a dialogue to jointly make decisions, with reciprocated sharing of information that both find beneficial to reach the best decision. SDM entails much more than patient education or informed consent: there must be bidirectional transfer of knowledge, discussion of patient preference, and a process of deliberation reaching consensus. Patient decision aids have been shown to improve patient understanding of options and risks, enhance the patient's involvement, and focus their comprehension of treatment preferences. Patient decision aids also may be of value in strengthening the physician-patient relationship. The need to emphasize SDM should be integrated into the quality process at every level to make it meaningful, not an apparently arbitrary obstacle that requires discovery of a shrewd work-around. A more patient-oriented consideration of the benefits of symptom relief and improved quality of life, in addition to survival and freedom from adverse events, could only be beneficial.

Advances in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Covalent Inhibition.

Peptidoglycan is a cross-linked polymer responsible for maintaining the bacterial cell wall integrity and morphology in Gram-negative and Gram-positive bacteria. The peptidoglycan pathway consists of the enzymatic reactions held in three steps: cytoplasmic, membrane-associated, and periplasmic. The Mur enzymes (MurA-MurF) are involved in a cytoplasmic stage. The UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme is responsible for transferring the enolpyruvate group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG) to form UDP-N-acetylglucosamine enolpyruvate (EP-UNAG). Fosfomycin is a natural product analogous to PEP that acts on the MurA target enzyme via binding covalently to the key cysteine residue in the active site. Similar to fosfomycin, other MurA covalent inhibitors have been described with a warhead in their structure that forms a covalent bond with the molecular target. In MurA, the nucleophilic thiolate of Cys115 is pointed as the main group involved in the warhead binding. Thus, in this minireview, we briefly describe the main recent advances in the design of MurA covalent inhibitors.