Journal Article Robotic Preparation of Tissue Specimens for TEM and Volume EM Get access Thomas E Strader, Thomas E Strader Heartland Biotech, Madison, WI, United StatesUniversity of Wisconsin-Madison, Madison, WI, United States Corresponding author: tom.strader@heartlandbiotech.com Search for other works by this author on: Oxford Academic Google Scholar Benjamin K August, Benjamin K August University of Wisconsin-Madison, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Ru-ching Hsia Ru-ching Hsia Carnegie Institute of Science, Baltimore, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 2089–2090, https://doi.org/10.1093/micmic/ozad067.1081 Published: 22 July 2023

The proinflammatory state associated with diabetes mellitus (DM) remains poorly understood. We found patients with DM have 3- to 14-fold elevations of blood-borne microparticles (MPs) that bind phalloidin (Ph; Ph positive [+] MPs), indicating the presence of F-actin on their surface. We hypothesized that F-actin-coated MPs were an unrecognized cause for DM-associated proinflammatory status. Ph+MPs, but not Ph-negative MPs, activate human and murine (Mus musculus) neutrophils through biophysical attributes of F-actin and membrane expression of phosphatidylserine (PS). Neutrophils respond to Ph+MPs via a linked membrane array, including the receptor for advanced glycation end products and CD36, PS-binding membrane receptors. These proteins in conjunction with TLR4 are coupled to NO synthase 1 adaptor protein (NOS1AP). Neutrophil activation occurs because of Ph+MPs causing elevations of NF-κB and Src kinase (SrcK) via a concurrent increased association of NO synthase 2 and SrcK with NOS1AP, resulting in SrcK S-nitrosylation. We conclude that NOS1AP links PS-binding receptors with intracellular regulatory proteins. Ph+MPs are alarmins present in normal human plasma and are increased in those with DM and especially those with DM and a lower-extremity ulcer.

Background Mild traumatic brain injury (mTBI) generally resolves within weeks. However, 15-30% of patients present persistent pathological and neurobehavioral sequelae that negatively affect their quality of life. Hyperhomocysteinemia (HHCY) is a neurotoxic condition derived from homocysteine accumulation above 15 μM. HHCY can occur in diverse stressful situations, including those sustained by U.S. active-duty service members on the battlefield or during routine combat practice. Mild-TBI accounts for more than 80% of all TBI cases, and HHCY exists in 5-7% of the general population. We recently reported that moderate HHCY exacerbates mTBI-induced cortical injury pathophysiology, including increased oxidative stress. Several studies have demonstrated hippocampus vulnerability to oxidative stress and its downstream effects on inflammation and cell death. Objective This study aimed to assess the deleterious impact of HHCY on mTBI-associated hippocampal pathological changes. We tested the hypothesis that moderate HHCY aggravates mTBI-induced hippocampal pathological changes. Methods HHCY was induced in adult male Sprague-Dawley rats with a high methionine dose. Rats were then subjected to mTBI by controlled cortical impact under sustained HHCY. Blood plasma was assessed for homocysteine levels and brain tissue for markers of oxidative stress, blood-brain barrier integrity, and cell death. Endothelial cell ultrastructure was assessed by Electron Microscopy and working memory performance using the Y maze test. Results HHCY increased the hippocampal expression of nitrotyrosine in astroglial cells and decreased tight junction protein occludin levels associated with the enlargement of the endothelial cell nucleus. Furthermore, HHCY altered the expression of apoptosis-regulating proteins α-ii spectrin hydrolysis, ERK1/2, and AKT phosphorylation, mirrored by exacerbated mTBI-related hippocampal neuronal loss and working memory deficits. Conclusion Our findings indicate that HHCY is an epigenetic factor that modulates mTBI pathological progression in the hippocampus and represents a putative therapeutic target for mitigating such physiological stressors that increase severity.

Background We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis -infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. Methods This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, GroEL, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. Results More C. trachomatis -infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9-40.7% of females vs . 24.2-30.0% of males), with statistical significance for PmpC, F and H (P<0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis -infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis (P<0.05) was observed. Conclusions Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs . urethra in males) stimuli control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum.

Abstract Background Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs. Methods Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively. Results As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival. Conclusion These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs.

Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1–SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1–SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1 +/− autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1–SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.

e21144 Background: Immune-checkpoint inhibitors (ICIs) have revolutionized the therapeutic landscape of lung cancer patients. However, its low treatment efficacy is still an issue and the current standard of care tissue PD-L1 presents high variability. Liquid biopsy is a promising tool in the discovery of biomarkers in body fluids. In particular, extracellular vesicles (EVs) can present PD-L1 in their membranes, playing a role in the inhibition of the anti-tumor immune response. Likewise, TGF-β is crucial in the immune response found in the circulation and into EVs. On the other hand, radiomics analysis of conventional imaging provides information about tumor heterogeneity and immune response. Hence, we aimed to evaluate the predictive role of circulating biomarkers in lung cancer patients undergoing ICIs and the additional value of radiomics data. Methods: This is a retrospective analysis of 30 advanced/metastatic non-small lung cancer patients treated with ICIs. Plasma samples were collected at baseline and at 8 weeks during treatment, matching the first response evaluation. Patients with complete, partial response, or stable disease were classified as responders and those with progressive disease as non-responders following RECIST v1.1. EVs were isolated from plasma by ultracentrifugation and PD-L1 expression was revealed by immunoblot. Circulating and EV levels of TGF-β were analyzed by ELISA. Additionally, 400 radiomics features from target and non-target lesions were analyzed to evaluate response in 24 patients according to RECIST v1.1 and irRECIST. Robustness of predictive models was validated by ridge penalty and leave-one-out cross-validation. Results: The analysis of the dynamics of EV PD-L1 during treatment identified increased levels in non-responders in comparison to responders ( p= 0.012), while tissue PD-L1 levels were not associated to the response ( p= 0.585). The predictive model for EV PD-L1 reported a high accuracy with an area under the curve (AUC) = 77%, 91.7% sensitivity, and 61.1% specificity. The combination with radiomics, improved the accuracy and reported an AUC = 83%, 82% sensitivity, and 77% specificity. Additionally, the analysis of the association of the circulating biomarkers with the outcome revealed that increasing dynamics of EV PD-L1 and high baseline EV TGF-β were associated with shorter progression-free survival and overall survival, outperforming the circulating levels of TGF-β. Conclusions: This pilot study demonstrated that EV PD-L1 could serve as better predictive factor than tissue PD-L1 for the stratification of lung cancer patients undergoing ICIs and that it could be complemented with radiomics. Moreover, EV levels of PD-L1 and TGF-β have potential for the stratification and prognosis of patients treated with ICIs and their novel combinations with TGF-β blockade.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

Mechanical forces are conducted through myofibers and into nuclei to regulate muscle development, hypertrophy, and homeostasis. We hypothesized that nuclei in aged muscle have changes in the nuclear envelope and associated proteins, resulting in altered markers of mechano-signaling. Methods: YAP/TAZ protein expression and gene expression of downstream targets, Ankrd1 and Cyr61, were evaluated as mechanotransduction indicators. Expression of proteins in the nuclear lamina and the nuclear pore complex (NPC) were assessed, and nuclear morphology was characterized by electron microscopy. Nuclear envelope permeability was assessed by uptake of 70 kDa fluorescent dextran. Results: Nuclear changes with aging included a relative decrease of lamin beta 1 and Nup107, and a relative increase in Nup93, which could underlie the aberrant nuclear morphology, increased nuclear leakiness, and elevated YAP/TAZ signaling. Conclusion: Aged muscles have hyperactive nuclear-cytoplasmic signaling, indicative of altered nuclear mechanotransduction. These data highlight a possible role for the nucleus in aging-related aberrant mechano-sensing.

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

The Electron Microscopy Core Imaging Facility (EMCIF) of the University of Maryland Baltimore conducts 100-150 electron microscopy (EM) -related research projects and processes a wide range of specimens each year. Many experimental procedures are customized to meet the demands of each specific research project. In order to minimize the time required for record-keeping of a large number of samples and experimental details, we have developed a management database using Microsoft Office Access, a program included in the Microsoft Office Professional Suite. EMCIF day-to-day records are divided into several main data tables including client information, project information, sample description, project charges, project experiments, and lab reagents. Records are entered using a custom-designed entry form with drop-down menus to minimize typing. The whole database is safely stored in a web-based server and can be accessed by all EMCIF personnel from their own computers simultaneously. Using built-in command buttons, documents such as invoices, quotes, project summaries and experimental workflows can be printed as reports, exported to other programs, or e-mailed to clients with a single click. This Do-It-Yourself management system was initially constructed in 2009 with five tables designed to manage service records. It has been expanded and modified through the years to incorporate new experimental procedures, reagents, as well as other critical information. The database recently celebrated its ten years anniversary. It currently includes records for approximately 10,000 EM specimens, 1,500 projects and over 500 clients. The ease of navigation of the database allows the EMCIF director and its staff to track down information concerning any client, project, or sample with a few clicks. A significant advantage of this 'DIY' database is its ease of adaptation and expansion to fit the changing needs of both the facility and its clients, an option often not available in commercial.

The commercial marketplace has seen a rapid increase in the number of over-the-counter charcoal-containing mouthwashes. The purpose of this systemic review was to examine the clinical and laboratory evidence supporting therapeutic claims of efficacy and safety of use of charcoal-based mouthwashes. Secondly, the product labels and information of 36 commercially marketed charcoal mouthwashes were reviewed for active ingredients. Only 8% of charcoal mouthwashes contained an active ingredient, such as cetylpyridinium chloride or chlorhexidine. There is insufficient evidence to substantiate the therapeutic and cosmetic marketing claims of charcoal-based mouthwashes, including antimicrobial activity, anti-halitosis, tooth whitening, periodontal disease control, caries reduction and tooth remineralisation, among others. Moreover, there is no available information on charcoal particulate size or abrasivity of any of these products. Dental clinicians should advise their patients to exercise caution when using over-the-counter charcoal-containing mouthwashes because of the lack of evidence supporting therapeutic or cosmetic effectiveness as well as safety.

“Giant” phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them “mottled capsids”. Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a “twisted hook” conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

Pancreatic cancer represents a life threatening disease with rising mortality. Although the synergistic combination of gemcitabine and albumin-bound paclitaxel has proven to enhance the median survival rates as compared to gemcitabine alone, their systemic and repeated co-administration has been associated with serious toxic side effects and poor patient compliance. For this purpose, we designed a thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the local and sustained delivery of gemcitabine (GEM) and paclitaxel (PTX) to pancreatic cancer. GEM and PTX were loaded into PR_b-functionalized liposomes targeting integrin α5β1, which was shown to be overexpressed in pancreatic cancer. PR_b is a fibronectin-mimetic peptide that binds to α5β1 with high affinity and specificity. The PR_b liposomes were encapsulated into a poly(δ-valerolactone-co-D,L-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-D,L-lactide) (PVLA-PEG-PVLA) hydrogel and demonstrated sustained release of both drugs compared to PR_b-functionalized liposomes free in solution or free drugs in the hydrogel. Moreover, the hydrogel-nanoparticle system was proven to be very efficient towards killing monolayers of human pancreatic cancer cells (PANC-1), and showed a significant reduction in the growth pattern of PANC-1 tumor spheroids as compared to hydrogels encapsulating non-targeted liposomes with GEM/PTX or free drugs, after a one week treatment period. Our hybrid hydrogel-nanoparticle system is a promising platform for the local and sustained delivery of GEM/PTX to pancreatic cancer, with the goal of maximizing the therapeutic efficacy of this synergistic drug cocktail while potentially minimizing toxic side effects and eliminating the need for repeated co-administration.

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHAT as the type strain.

An abstract is not available for this content so a preview has been provided. As you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

Lipid droplets (LDs) are dynamic organelles that interact with various cell components such as the endoplasmic reticulum, endosomes and mitochondria. Excessive or defective LD accumulation are implicated in obesity and other metabolic diseases such as diabetes and atherosclerosis [1]. We describe here our investigation of the role of LDs in obesity and their interactions with other organelles using transgenic mice over-expressing perilipin 5 under a α-myosin heavy chain promoter [2].

An abstract is not available for this content so a preview has been provided. As you have access to this content, a full PDF is available via the ‘Save PDF’ action button.