A workflow to practically apply true dose considerations to in vitro testing for Next Generation Risk Assessment.

With the move away from safety testing assessment based on data generated in experimental animals the concept of Next Generation Risk Assessment (NGRA) has arisen which instead uses data from in silico and in vitro models. A key uncertainty in risk assessment is the actual dose of test chemical at the target site, and therefore surrogate dose metrics, such as nominal concentration in test media are used to describe in vitro effect (or no-effect) doses. The reliability and accuracy of the risk assessment therefore depends largely on our ability to understand and characterise the relationship between the dose metrics used and the actual biologically effective dose at the target site. The objective of this publication is to use 40 case study chemicals to illustrate how in vitro dose considerations can be applied to characterise the "true dose" and build confidence in the understanding of the biologically effective dose in in vitro test systems for the determination e.g. points of departure (PoDs) for NGRA. We propose a workflow that can be applied to assess whether the nominal test concentration can be considered a conservative dose metric for use in NGRA. The workflow examines the implications of volatility, stability, hydrophobicity, binding to plastic and serum, solubility, and the potential use of in silico models for some of these parameters. For the majority of the case study chemicals we found that the use of nominal concentrations in risk assessment would result in conservative decision making. However, for serval chemicals a potential for underestimation of the risk in humans in vivo based on in vitro nominal effect concentrations was identified, and approaches for refinement by characterisation of the actual effect concentration are proposed.

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification. The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Conformational ensemble of yeast ATP synthase at low pH reveals unique intermediates and plasticity in F1-Fo coupling.

Mitochondrial adenosine triphosphate (ATP) synthase uses the proton gradient across the inner mitochondrial membrane to synthesize ATP. Structural and single molecule studies conducted mostly at neutral or basic pH have provided details of the reaction mechanism of ATP synthesis. However, pH of the mitochondrial matrix is slightly acidic during hypoxia and pH-dependent conformational changes in the ATP synthase have been reported. Here we use single-particle cryo-EM to analyze the conformational ensemble of the yeast (Saccharomyces cerevisiae) ATP synthase at pH 6. Of the four conformations resolved in this study, three are reaction intermediates. In addition to canonical catalytic dwell and binding dwell structures, we identify two unique conformations with nearly identical positions of the central rotor but different catalytic site conformations. These structures provide new insights into the catalytic mechanism of the ATP synthase and highlight elastic coupling between the catalytic and proton translocating domains.

In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.

Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Comparison of three different approaches in treatment of mandibular condylar fractures - Our experience.

Background: The principal transfacial routes described to approach condylar process fractures are preauricular, retromandibular, high submandibular, and their various modifications. The selection of a specific surgical approach mainly depends on the level of condylar fracture, displacement/dislocation of the proximal segment and time elapsed from the date of trauma. Not too many studies proclaim a unanimous consensus on "gold standard" approaches for particular levels of fracture. Aim: This study was conducted with the aim to lay down guidelines for determining the ideal surgical approach for treating different condylar fractures based on different clinical situations. Methodology: This prospective study was conducted on 60 patients that underwent open reduction internal fixation of condylar fractures according to preset study design. Inclusion criteria were fracture displacement more than 10 degrees, dislocation, shortening of ramal height more than 2 mm. Patients having maxillofacial fractures other than mandible and condylar fractures in edentulous patients were excluded from this study. Results: The risk of facial nerve injury was more in endaural group and parotid fistula was more evident in retromandibular subparotid group but there was no statistical difference between the groups. The surgical scar was inconspicuous in all three groups. Excellent results were achieved in the other parameters such as mouth opening, range of motion, bite force, and occlusion with all the three approaches. Postoperative CT scan showed satisfactory anatomical reduction. Conclusion: This study concludes that the decision-making on the approach to be selected for any particular condylar fracture depends on the level of fracture, presence of fracture dislocation, and time elapsed from the date of trauma.

Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Comparative evaluation of histopathological analysis, KOH wet mount and fungal culture to diagnose fungal infections in post-COVID patients.

Context and Aim: There is increasing prevalence of post-COVID fungal infection of rhinoorbitocerebral region especially mucormycosis and aspergillosis in India.[1] Early diagnosis of these fungal infections are of utmost importance, since it may improve outcome and survival.[2],[3],[4],[5],[6],[7],[8] The objective of this study was to evaluate and compare routine laboratory diagnostic methods, that is, histopathological examination, KOH wet mount and fungal culture in the diagnosis of post-COVID fungal infections. Materials and Methods: A total of 106 specimens of clinically suspected patients of post-COVID fungal infection of rhinoorbitocerebral region received in histopathology department were included in this study. The data of KOH wet mount and culture were acquired from the microbiology department after histopathological examination. Result: Approximately 88.68% of patients were diagnosed having fungal infections by one of the laboratory methods. The sensitivity of histopathological examination was highest (79.78%), followed by KOH wet mount (58.51%) and fungal culture (35.10%). Rhizopus species of zygomycetes group were the most common isolate (24.24%) on SDA culture. Overall 76% concordance was found between histopathological examination and fungal culture report for morphological identification of fungi. Conclusion: For the diagnosis of post-COVID fungal infection of Rhino-orbito-cerebral region, histopathological examination is was found to be more sensitive and rapid method to detect fungal hyphae. It leads to early treatment, prevents morbidity and mortality.

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.

Local Monomer Levels and Established Filaments Potentiate Non-Muscle Myosin 2 Assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

Extraction Solvents Affect Anthocyanin Yield, Color, and Profile of Strawberries.

Anthocyanins are a major group of plant pigments that have antioxidant activities. Pigments play a major role in human health and have attracted a lot of attention globally. Many factors affect anthocyanin yields, such as solvent type, incubation time, solvent-to-sample ratio, sample type, and temperature. The first parameter was tested, and the rest were considered constant in this experiment. A total of nine organic and water-based solvents (methanol and chloroform: methanol, acetone, ethanol, water) and their combinations were compared to extract anthocyanins from freshly-pureed strawberries. Solvents changed anthocyanin yield, color parameters, and profile. The color parameters of a* values lower than 30, L* values higher than 85, hue angle more than 40, and chroma less than 30 indicated some color degradation in strawberry anthocyanins. Therefore, the best solvents for anthocyanin assessment were methanol and methanol: water. The second-best solvent was the pH differential buffers. Other solvents such as ethanol, chloroform: methanol, water, and water-based solvents extracted considerable amounts of anthocyanins; however, they showed some degree of color degradation, evidenced by the color parameters. Acetone did not yield a stable extract which degraded over 48 h of storage at 4 °C. The extraction solvent determined the main anthocyanin of the anthocyanins profile. Pelargonidin was the major anthocyanin in chloroform: methanol solvent, while delphinidin was dominant in all other solvents.

Arthrocentesis Versus Level 1 Arthroscopy in Internal Derangement of Temporomandibular Joint.

Introduction: Internal derangement of the temporomandibular joint is defined as an abnormal position of the articular disc in relation with mandibular condyle and articular eminence presenting as disc displacement with or without reduction. Methodology: This study was conducted on thirty patients diagnosed with Internal derangement of TMJ consisting of 8 males and 22 females averaging 34.6 years. Two groups Conventional Arthrocentesis (Group A) and Level 1 Arthroscopy (Group B) consisted of 15 cases each divided alternately. Clinical evaluation parameters included VAS for pain, maximal interincisal opening, deviation on mouth opening, range of motion including lateral excursion & protrusion movements recorded at 1 week, 1 month & 6 months postoperatively. Wilke's Staging according to MRI findings was recorded preoperatively and 6 months postoperatively. Results: At 6 month follow-up, average reduction in VAS for pain & deviation on mouth opening was 72.43% & 24.73% in Group A and 77.66% & 65.41% in Group B, respectively. Average increase in MIO, right & left excursion & protrusion movements was 29.55%, 31.33%, 20.12% & 32.45% in Group A and 34.94%, 41.37%, 39.29% and 36.51% in Group B, respectively. Improved results were obtained clinically for all Wilke's stages in both groups with more number of patients improving in Group B. Conclusion: On comparing results, improvement was observed in various clinical evaluation parameters of both the groups at 6 months follow-up. However, statistically significant & better results were obtained for the Arthroscopy group.

Odynophagia in a young adult: revisiting herpetic esophagitis and eosinophilic esophagitis.

An immunocompetent man in his 20s presented with a 24-hour history of severe odynophagia, nausea, vomiting and throat pain. Esophagogastroduodenoscopy (EGD) revealed severe esophagitis with ulcerated mucosa, exudative debris, haemorrhage and multiple erosions. Biopsy of the oesophageal tissue demonstrated marginated chromatin, multinucleated giant cells and molding of nuclei, consistent with herpes simplex virus esophagitis (HSE). Treatment with oral acyclovir led to the complete resolution of symptoms. The patient subsequently developed dysphagia again, 8 months later. EGD showed furrowing and concentric rings typical of eosinophilic esophagitis (EoE), a diagnosis confirmed by biopsy. Treatment with a proton pump inhibitor and swallowed topical corticosteroids led to symptomatic improvement. Thus, HSE can occur in immunocompetent hosts and on occasion, HSE may be a harbinger of EoE, as evidenced by our extensive literature review. Mechanical disruption of the mucosal barrier by viruses, facilitating food allergen penetration, and associated immunological signaling abnormalities may be responsible phenomena requiring further elucidation.

Pseudomonas aeruginosa persister cell formation upon antibiotic exposure in planktonic and biofilm state.

Persister cell (PC) is dormant, tolerant to antibiotics, and a transient reversible phenotype. These phenotypes are observed in P. aeruginosa and cause bacterial chronic infection as well as recurrence of biofilm-mediated infection. PC formation requires stringent response and toxin-antitoxin (TA) modules. This study shows the P. aeruginosa PC formation in planktonic and biofilm stages on ceftazidime, gentamicin, and ciprofloxacin treatments. The PC formation was studied using persister assay, flow cytometry using Redox Sensor Green, fluorescence as well as Confocal Laser Scanning Microscopy, and gene expression of stringent response and TA genes. In the planktonic stage, ceftazidime showed a high survival fraction, high redox activity, and elongation of cells was observed followed by ciprofloxacin and gentamicin treatment having redox activity and rod-shaped cells. The gene expression of stringent response and TA genes were upregulated on gentamicin followed by ceftazidime treatment and varied among the isolates. In the biofilm stage, gentamicin and ciprofloxacin showed the biphasic killing pattern, redox activity, gene expression level of stringent response and TA varied across the isolates. Ceftazidime treatment showed higher persister cells in planktonic growth while all three antibiotics were able to induce persister cell formation in the biofilm stage.

Comparing pH differential and methanol-based methods for anthocyanin assessments of strawberries.

Anthocyanins are a group of water-soluble polyphenolic pigments found primarily in flowers, vegetables, and fruits. These pigments play critical roles in plant and human health. Spectrophotometric methods are a simple and inexpensive way to quantify anthocyanins in plant tissues. Two main spectrophotometric methods have been developed, organic solvent-based, and pH differential methods. Both of these methods are subject to interference from light-absorbing impurities and need to be optimized for different matrixes of different plant materials. Eight methods have been tested in this experiment to quantify anthocyanins in strawberry fruits. Six organic solvent-based methods tested methanol, chloroform-methanol, and MgO in different ratios. The other two methods were pH differential method and a combination of organic solvent-based and pH differential method. Two methods used organic solvents (methanol and chloroform-methanol) were the best in extracting anthocyanin from strawberry fruits. Adding MgO increased the pH of the extract and was less efficient in anthocyanin extraction. All other methods had lower anthocyanin yield compared with methanol and chloroform-methanol methods and are not recommended for strawberry fruit anthocyanin extraction.

Are Non-animal Systemic Safety Assessments Protective? A Toolbox and Workflow.

An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions. The toolbox includes physiologically based kinetic (PBK) models to estimate systemic Cmax levels in humans, and 3 bioactivity platforms, comprising high-throughput transcriptomics, a cell stress panel, and in vitro pharmacological profiling, from which points of departure are estimated. A Bayesian model was developed to quantify the uncertainty in the Cmax estimates depending on how the PBK models were parameterized. The feasibility of the evaluation approach was tested using 24 exposure scenarios from 10 chemicals, some of which would be considered high risk from a consumer goods perspective (eg, drugs that are systemically bioactive) and some low risk (eg, existing food or cosmetic ingredients). Using novel protectiveness and utility metrics, it was shown that up to 69% (9/13) of the low risk scenarios could be identified as such using the toolbox, whilst being protective against all (5/5) the high-risk ones. The results demonstrated how robust safety decisions could be made without using animal data. This work will enable a full evaluation to assess how protective and useful the toolbox and workflow are across a broader range of chemical-exposure scenarios.

Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease.

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.