A rare cutaneous infection with Mycobacterium chelonae in a pediatric patient treated with trametinib for KRAS-mutated diffuse glioma.

Mitogen-activated extracellular signal-regulated kinase inhibitors (MEKi) represent a promising new therapy for pediatric patients with low-grade gliomas, which frequently have abnormal signaling within the mitogen-activated protein kinase (MAP kinase) pathway. However, understanding of long-term efficacy and toxicity is limited in pediatric glioma patients. This article describes a rare presentation of a widespread cutaneous infection with Mycobacterium chelonae in a pediatric patient with a low-grade glioma treated with trametinib.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

First confirmed case of equine pythiosis in Northern Veracruz, Mexico.

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico.

Methods comparison of two-dimensional gel electrophoresis for host cell protein characterization.

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non-traditional method of 2D-DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP-based applications.

Lipoprotein Glomerulopathy With Complete Resolution With Fenofibrate: Report of First Case From Pakistan.

Background: Lipoprotein glomerulopathy is an infrequent glomerular disorder that culminates in nephrotic syndrome and often progresses to kidney failure. Whereas most patients have been reported in Japan and China, limited reports have been documented outside these regions. This patient represents the first report of lipoprotein glomerulopathy in Pakistan. Case Presentation: A 25-year-old male patient, hypertensive for 2 years, presented with progressive body edema, frothy urine, and fatigue. Examination revealed elevated blood pressure, bilateral pedal edema, and positive shifting dullness. Laboratory results showed significant proteinuria and elevated cholesterol and triglyceride levels. Renal biopsy revealed enlarged glomeruli with a dilated capillary lumen filled with pale-staining mesh-like material "lipoprotein thrombi." Mild tubular atrophy and interstitial inflammation were observed. No interstitial fibrosis was evident. Electron microscopy detailed the lipoprotein thrombi with lipid granules and vacuoles of various sizes. A diagnosis of lipoprotein glomerulopathy was rendered. Treatment with fenofibrate, rosuvastatin, and captopril led to notable improvements in symptoms, blood pressure, and lipid levels during a 6-month follow-up. Subsequent biopsy showed complete resolution of the lipoprotein thrombi and a significant reduction in subendothelial granular densities. However, the flocculent subendothelial material persisted to some extent despite the complete resolution of lipoprotein thrombi. Conclusion: This report underscores the rarity of lipoprotein glomerulopathy in Pakistan and contributes valuable insights into its histopathologic features and global epidemiology. This unique instance aims to raise awareness among healthcare professionals, aiding in improved recognition of this rare entity. The favorable response to fenofibrate treatment underscores its effectiveness in managing lipoprotein glomerulopathy.

Characterization of Klebsiella pneumoniae Extracellular Polysaccharides.

Klebsiella pneumoniae is a clinically significant, Gram-negative pathogen in which the production of extracellular polysaccharides is a key virulence factor. Extracellular polysaccharides such as the capsule and its mucoviscosity play a significant role in K. pneumoniae infection. In this article, we explain several standard protocols used to characterize the extracellular polysaccharides of K. pneumoniae. Several of these protocols are adapted specifically for K. pneumoniae and describe methods to purify and quantify the extracellular polysaccharide of K. pneumoniae. We also present a standardized protocol to quantify K. pneumoniae mucoviscosity, a unique feature of K. pneumoniae extracellular polysaccharide. These protocols will help create uniformity in standard protocols used in K. pneumoniae extracellular polysaccharide studies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extracellular polysaccharide isolation and purification Basic Protocol 2: Large-scale isolation and purification of extracellular polysaccharide Basic Protocol 3: Uronic acid quantification of extracellular polysaccharide Basic Protocol 4: Extracellular polysaccharide visualization by SDS-PAGE Basic Protocol 5: Klebsiella pneumoniae mucoviscosity measurement by sedimentation resistance assay Alternate Protocol 5: 96-well plate-based Klebsiella pneumoniae sedimentation resistance assay Support Protocol 5: Determination of plate to cuvette conversion factor.

Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

Comparative Study of Time-Dependent Aluminum Exposure and Post-Exposure Recovery Shows Better Improvement in Synaptic Changes and Neuronal Pathology in Rat Brain After Short-Term Exposure.

Aluminum is a ubiquitous metal that causes multiple brain pathologies such as, cognitive dysfunction and Alzheimer's disease like symptoms. Exposure to aluminum through drinking water is responsible for hampering learning and memory. This study aimed to compare (1) the time-dependent effect of aluminum exposure (keeping total exposure of 5850 mg/kg same) in two durations, 30 and 45 days, and (2) to compare post-exposure self-recovery effect after 20 days in both (30 and 45 days exposure) groups. Rats were given 130 and 195 mg/kg of AlCl3·6H2O for 45 and 30 days respectively, to see the time-dependent exposure effect. At the end of exposure, rats were given distilled water and allowed to self-recover for 20 days to study the recovery. Expression levels of synaptic genes (Syp, SNAP25, Nrxn1/2, PSD95, Shank1/2, Homer1, CamkIV, Nrg1/2 and Kalrn) were measured using qPCR and compared in the exposure and recovery groups. Cellular morphology of the rat brain cortex and hippocampus was also investigated. Damage in lipid and protein profile was measured by employing FTIR. Results showed downregulation of mRNA expression of synaptic genes, plaques deposition, disorganization in lipid and protein profile by increasing membrane fluidity, and disorder and alteration of protein secondary structure after both exposure periods. However, better improvement/recovery in these parameters were observed in recovery group of 30 days aluminum exposure compared to 45 days aluminum exposure group. Taken together, these results suggested that short-term exposure resulted in better restoration of lipid and protein profile after time-dependent exposure of aluminum than prolonged exposure.

Native valve Aspergillus fumigatus endocarditis in a patient with autoimmune hepatitis on low dose systemic corticosteroids: A case report.

Aspergillus infectious endocarditis (IE) is a rare cause of culture-negative endocarditis. The main risk factors are severe immunosuppression and prosthetic heart valve. We describe a case of Aspergillus fumigatus IE on a native mitral valve in a patient with autoimmune hepatitis in remission while on low dose corticosteroids. The case is unique due to the patients' low initial risk for invasive fungal disease, its clinical presentation and successful management with emergency surgery and antifungal therapy. After literature review we have not found a similar case report. The patient presented with right-sided eyesight deterioration due to endophthalmitis. Vitrectomy was performed and Aspergillus fumigatus grew on culture. IE on a native mitral valve was confirmed with echocardiography. The patient developed signs of acute heart failure soon after hospital admission and was diagnosed with several septic emboli (kidney, spleen, thumb, right common femoral artery). He was initially treated with surgical valve replacement, dual antifungal therapy with liposomal amphotericin B (LAmB) and voriconazole and vitrectomy, including intravitreal amphotericin B application. Long-term triazole therapy was not possible due to hepatotoxicity. The patient was maintained on intermittent LAmB for 12 months and is without signs of recurrence ten months after therapy was discontinued. Aspergillus can cause invasive infection in patients with autoimmune hepatitis on low dose corticosteroids. Early diagnosis followed by emergency surgical valve replacement and systemic antifungal therapy can improve prognosis. Additional studies are needed to evaluate alternative methods and duration of antimicrobial therapy following Aspergillus IE.

Persistent white matter vulnerability in a mouse model of mild traumatic brain injury.

BACKGROUND: Following one mild traumatic brain injury (mTBI), there is a window of vulnerability during which subsequent mTBIs can cause substantially exacerbated impairments. Currently, there are no known methods to monitor, shorten or mitigate this window. METHODS: To characterize a preclinical model of this window of vulnerability, we first gave male and female mice one or two high-depth or low-depth mTBIs separated by 1, 7, or 14 days. We assessed brain white matter integrity using silver staining within the corpus callosum and optic tracts, as well as behavioural performance on the Y-maze test and visual cliff test. RESULTS: The injuries resulted in windows of white matter vulnerability longer than 2 weeks but produced no behavioural impairments. Notably, this window duration is substantially longer than those reported in any previous preclinical vulnerability study, despite our injury model likely being milder than the ones used in those studies. We also found that sex and impact depth differentially influenced white matter integrity in different white matter regions. CONCLUSIONS: These results suggest that the experimental window of vulnerability following mTBI may be longer than previously reported. Additionally, this work highlights the value of including white matter damage, sex, and replicable injury models for the study of post-mTBI vulnerability and establishes important groundwork for the investigation of potential vulnerability mechanisms, biomarkers, and therapies.

Time Course of Remote Neuropathology Following Diffuse Traumatic Brain Injury in the Male Rat.

Traumatic brain injury (TBI) can affect different regions throughout the brain. Regions near the site of impact are the most vulnerable to injury. However, damage to distal regions occurs. We investigated progressive neuropathology in the dorsal hippocampus (near the impact) and cerebellum (distal to the impact) after diffuse TBI. Adult male rats were subjected to midline fluid percussion injury or sham injury. Brain tissue was stained by the amino cupric silver stain. Neuropathology was quantified in sub-regions of the dorsal hippocampus at 1, 7, and 28 days post-injury (DPI) and coronal cerebellar sections at 1, 2, and 7 DPI. The highest observed neuropathology in the dentate gyrus occurred at 7 DPI which attenuated by 28 DPI, whereas the highest observed neuropathology was at 1 DPI in the CA3 region. There was no significant neuropathology in the CA1 region at any time point. Neuropathology was increased at 7 DPI in the cerebellum compared to shams and stripes of pathology were observed in the molecular layer perpendicular to the cerebellar cortical surface. Together these data show that diffuse TBI can result in neuropathology across the brain. By describing the time course of pathology in response to TBI, it is possible to build the temporal profile of disease progression.

In Vivo Biofilm Formation of Pathogenic Leptospira spp. in the Vitreous Humor of Horses with Recurrent Uveitis.

Equine recurrent uveitis (ERU) causes painful inflammatory attacks and oftentimes blindness in the affected eyes. The disease is considered a late sequela of systemic leptospirosis. The most effective therapy is the surgical removal of the vitreous (vitrectomy), which is not only therapeutic, but provides vitreous material that can be assessed diagnostically. For example, the lipL32 gene, culturable Leptospira spp., and anti-Leptospira antibodies have all been detected in vitreous samples obtained from eyes with chronic ERU. Despite this clear evidence of leptospiral involvement, the systemic administration of antibiotics in infected horses is ineffective at resolving ERU. This syndrome of chronic recurrent inflammation, which is unresponsive to antibiotic therapy, combined with apparent bacteria evading the immune response, is consistent with a biofilm-associated infection. The purpose of this study, therefore, was to detect the in vivo biofilm formation of Leptospira spp. in vitreous samples collected during vitrectomy and examined using a Warthin-Starry silver stain and immunohistochemistry. All known steps of biofilm formation were visualized in these samples, including individual Leptospira spp., leptospiral microcolonies and dense roundish accumulations of Leptospira spp. In many instances spirochetes were surrounded by an extracellular substance. Taken together, data from the present study show that ERU is a biofilm-associated intraocular leptospiral infection, which best explains the typical clinical course.

Alzheimer-like cell death after vanadium pentoxide inhalation.

Vanadium (V) toxicity depends on its oxidation state; it seems that vanadium pentoxide (V2O5) is the most toxic to the living cells. It has been reported that oral administration induces changes in motor activity and learning; in rats, I.P. administration increases lipid peroxidation levels in the cerebellum and the concentration of free radicals in the hippocampus and cerebellum. Mice that inhaled V2O5 presented a reduced number of tubulin+ in Leydig and Sertoli cells; it has also been reported that inhaled V2O5 induces loss of dendritic spines, necrosis, and hippocampus neuropil alterations; considering the direct consequence of the interaction of V with cytoskeletal components, makes us believe that V2O5 exposure could cause neuronal death in the hippocampus similar to that seen in Alzheimer disease. This work aimed to determine pyramidal hippocampal CA1 cytoskeletal alterations with Bielschowsky stain in rats exposed to V2O5. Male Wistar rats inhaled 0.02 M of V2O5 one h two times a week for two and six months. We found that rats, which inhaled V2O5 reached 56,57% of dead neurons after six months of inhalation; we recognize strong argyrophilic and collapsed somas and typical flame-shaped in all V-exposed rats hippocampus CA1 compared to controls. We also observe somatodendritic distortions. Axons and dendrites displayed thick dark bands replaced by noticeable thickening and nodosities and the cytoskeleton fibrillary proteins' linear traces. Our findings suggest that V2O5 inhalation induces Alzheimer-like cell death with evident cytoskeletal alterations.

Novel SOD1 monoclonal antibodies against the electrostatic loop preferentially detect misfolded SOD1 aggregates.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to motor neuron degeneration and paralysis. Superoxide dismutase (SOD1) mutations are the second most common cause of familial ALS and are responsible for up to 20 % of familial ALS cases. In ALS patients, SOD1 can form toxic misfolded aggregates that deposit in the brain and spinal cord. To better detect SOD1 aggregates and expand the repertoire of conformational SOD1 antibodies, SOD1 monoclonal antibodies were generated by immunizing SOD1 knockout mice with an SOD1 fragment consisting of amino acids 129-146, which make up part of the electrostatic loop. A series of hybridomas secreting antibodies were screened and five different SOD1 monoclonal antibodies (2C10, 2F8, 4B11, 5H5, and 5A10) were found to preferentially detect denatured or aggregated SOD1 by enzyme-linked immunosorbent assay (ELISA), filter trap assay, and immunohistochemical analysis in SOD1 mouse models. The staining with these antibodies was compared to Campbell-Switzer argyrophilic reactivity of pathological inclusions. These new conformational selective SOD1 antibodies will be useful for clinical diagnosis of SOD1 ALS and potentially for passive immunotherapy.

Re-evaluation of the 18 non-human protein standards used to create the empirical statistical model for decoy library searching.

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification.

A multiple signal amplification sandwich-type SERS biosensor for femtomolar detection of miRNA.

MicroRNAs are widely used as tumor markers for cancer diagnosis and prognosis. Herein, a multiple signal amplification sandwich-type SERS biosensor for femtomolar detection of miRNA is reported. The signal unit consisted of giant Au vesicles, DNA sequences and deposited silver nanoparticles. The giant Au vesicles provided large-volume hot spots because of sharp tips and abundant hotspot gaps, thus enhancing the electromagnetic intensity for the SERS performance. Further silver stain would easily lead to second-stage amplification of Raman signal. In addition, more SERS signal molecules R6G adsorbed on the signal unit with the aid of HCR and the controlled nanogaps between adjacent AgNPs, brought about the third-stage amplification. The capture unit, prepared by immobilizing the capture probe (CP) on the Fe3O4@AuNPs, could easily capture target miRNA and greatly simplify the separation step to improve reproducibility. The higher concentration of target miRNA definitely formed more sandwich-type structures with combination of capture unit and signal unit, resulting in multiple amplification of SERS signals. The proposed multiple signal amplification sandwich-type SERS biosensor could detect miRNA-141 at the femtomolar level with a low detection limit of 0.03 fM. Meanwhile, it exhibited high selectivity and accuracy, even for practical analysis in human serum. Therefore, the designed multiple signal amplification sandwich-type SERS biosensor would be a very promising alternative tool for the detection of miRNA and analogs in the field of biomedical diagnosis.

Histochemical and immunohistochemical detection of pathogenic leptospires serovars in tissues of some captive wildlife from a University zoo in Nigeria.

The detection and documentation of pathogenic Leptospira serovars in wild captive and zoological garden animals are scarce in literature from Nigeria. The knowledge of the prevalence of prevalence of pathogenic Leptospira serovars in these animals as a zoonotic risk to workers, zoo visitors and the general public is essential. This investigation was carried out on archival kidney and liver samples of captive and Zoological Garden animals (66) of an institutional facility, submitted for necropsy to the Department of Veterinary Pathology between the periods of 2010-2015. The gross diagnosis reports were obtained from the necropsy records, detection of pathogenic Leptospira serovars was by Warthin Starry silver staining and immunohistochemistry techniques using standard methods. Six samples out of the sixty-six samples were positive for leptospira four samples were positive by silver stain method, while two samples were positive by immunohistochemistry. In this study, serovar Pomona and grippotyphosa were detected in the foxes while serovar Pomona was detected in the horse. This study has revealed the presence of pathogenic leptospires in some captive wild and zoological garden animals.

Utility of Histologic and Histochemical Screening for 16S Ribosomal RNA Gene Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue for Bacterial Endocarditis.

OBJECTIVES: 16S ribosomal RNA (rRNA) sequencing is a powerful but expensive tool for the identification of bacteria in culture-negative endocarditis. Histologic criteria to screen formalin-fixed, paraffin-embedded (FFPE) specimens for testing are evaluated. METHODS: Sixty-eight cases of infective endocarditis and controls were histologically reviewed and analyzed by 16S rRNA gene sequencing. RESULTS: Sequencing identified a specific pathogenic organism in 33 (49%) of 68 cases with acute inflammation and in 0 of 10 controls (P = .004). Visualization of organisms by Gram or Grocott methenamine silver stains had the strongest association with positive sequencing, while antibiotic treatment effect and acid decalcification decreased sensitivity. Molecular identifications were concordant with blood culture results in 90% of the cases, and a positive sequencing result was obtained in approximately half of the cases with negative valve cultures. CONCLUSIONS: Histologic screening criteria are extremely helpful for identifying cases likely to be positive by molecular testing and can provide significant cost savings in filtering out low-yield specimens.

Disseminated Histoplasmosis presenting as oropharyngeal mass lesion.

A 62 year old man from Silchar in North East India presented with complaints of painful swallowing, hoarseness, fever, anorexia and weight loss. Oropharyngeal examination revealed reddish ulcero-nodular lesion involving left tonsillar area and base of tongue which was clinically suspicious of malignancy. Radiological examination revealed involvement of bilateral adrenals by a mass. The biopsy of the oropharyngeal lesion showed many fungal spores morphologically favoring Histoplasmosis. Treatment with Amphotericin B followed by Itraconazole resulted recovery of lesions.