Light Microscopy Research Articles

Macrocheles muscaedomesticae (Acari: Macrochelidae) associated with Stomoxys calcitrans (Diptera: Muscidae) in the municipality of Sabanalarga, Antioquia

Objective: To identify mites in Diptera in a wooded area of Sabanalarga, Department of Antioquia, Colombia. Materials and methods: Two "stable flies" (Stomoxys calcitrans) were collected in a corral used temporarily to move cattle located in a wooded area of the municipality of Sabanalarga, Antioquia-Colombia. The flies were stored in Eppendorf tubes containing 70% ethyl alcohol for further processing. In the laboratory, the flies were identified, and then the mites they carried on their ventral area were removed and counted. Subsequently, the mites were stored in 70% ethyl alcohol to be rinsed on slides and identified under a light microscope. Results: A total of 15 specimens of a mite of the order Mesostigmata, family Macrochelidae of the species Macrocheles muscaedomesticae, parasitizing the flies S. calcitrans, were identified. Conclusions: This is the first report on the parasitic association between phoretic mites Macrocheles muscaedomesticae and stable flies Stomoxys calcitrans in Sabanalarga, Antioquia-Colombia.

Determination of prevalence, risk factors and symptoms of urinary schistosomiasis among school children in Kaduna South Local Government Area, Kaduna State

Schistosomiasis negatively impacts children’s health. Children expose themselves daily to schistosome infections due to lack of awareness and knowledge about the disease or the danger of infested water bodies. This study was aimed at determining the prevalence, risk factors and symptoms of urinary schistosomiasis among school children in Kaduna South Local Government Area, Kaduna State, Nigeria. Four hundred (400) consented school children submitted 10mL urine each and responded to structured questionnaires. Sediment of each urine sample was examined by light microscopy, after centrifugation at 3000 rpm for 5 min. Overall prevalence of urinary schistosomiasis was 4.5%. Both male and female children were equally infected (4.5% each). Older children between 13-17 years old were more infected (5.0%) and more at risk (OR = 2.982) than younger children (8-12 years old) who had 1.7% of the infection. No infection was recorded among children who had awareness about the disease. Water-based activities that served as significant risk factors for the infection included swimming in river (8.7%, P=0.016, OR= 3.064), fishing (12.2%, P=0.005, OR=3.942) and washing of clothes in river (17.4%, P=0.002, OR=5.459). Significant symptoms of urinary schistosomiasis identified were frequent urination (12.5%, P=0.023, OR=3.612), abdominal pain (10.0%, P=0.045, OR=2.880), and terminal haematuria (13.6%, P=0.002, OR=4.526). Infected individuals were more at risk of experiencing pains during urination (7.4%, OR=2.047). Unawareness promotes the spread of schistosomiasis among Nigerian children; their continuous exposure to infested water poses a greater risk. Creation of awareness amongst children will help to control their play habits in unsafe water bodies.

Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

Prevalence of urinary schistosomiasis among school children in Giwa and Markafi Local Government Areas, Kaduna State, Nigeria

Urinary schistosomiasis is a neglected tropical disease that is affecting the health of many children, especially in Nigeria. Awareness about schistosomiasis in some communities is low; hence more children undertake activities in unsafe bodies of water. This study determined the prevalence, socio-demography, risk factors and symptoms of urinary schistosomiasis among school children in Giwa and Makarfi LGAs. A total of 200 school children were enrolled in this study and each of them submitted 10 mL urine sample. Each urine sample was concentrated by centrifugation and the sediment was examined using a light compound microscope. Data collected were analyzed using IBM SPSS Version 23 at 95% confidence interval. Overall prevalence of urinary schistosomiasis in this study was 19(9.5%). Spatial prevalence was 10.0% and 9.0% in Giwa and Makarfi LGAs respectively. There was no infection among the females (0.0%), but 13.5% of infection occurred among the males (P=0.003, OR =1.156). School children within the age-group of 18–19 years old had highest infection of 20.0%, followed by 12.1% and 5.6% among those within age-groups of 14-15 and 12-13 years old respectively. The infection was more occurring among children in junior secondary school (10.4%) than among those in senior secondary school (7.7%). Significant risk factors were irrigation farming, swimming, fishing and washing of clothes in rivers (P<0.05, OR >1). Significant symptoms of the disease among them included painful urination and terminal haematuria (P<0.05, OR >1). However, infected individuals mostly encountered frequent urination and abdominal pains (OR>1). Urinary schistosomiasis prevails in the study area. It is important to create widespread awareness and treatment intervention.

A new Actinella species (Eunotiaceae, Bacillariophyta) from the Yangambi Biosphere Reserve, Democratic Republic of the Congo

Background and aims – Actinella species from Central Africa are only rarely reported. The acid waters from many stream and small rivers in the Congo Basin form a potential environment for species of this genus. We studied several samples from the region of Yangambi to confirm whether species belonging to this genus are present. Material and methods – Samples for diatom investigation were collected in several streams and small rivers (Moni, Ngima, and Libongo), in the tropical rainforest in the Yangambi Biosphere Reserve, Tshopo Province, Democratic Republic of the Congo. The samples were prepared to obtain permanent microscope slides for light microscopy studies using standard methods and cleaned material for scanning electron microscopy. Key results – A novel Actinella was observed in the studied samples. The taxon was compared to other species such as A. disjuncta, A. lange-bertalotii, A. modesta, A. pereunotioides, and A. pseudohantzschia, although the new species morphologically resembles most A. eunotioides, a species discovered in the Amazon basin in Brazil, and A. eunotioides var. minor, described in 1966 from the Central African Republic. The taxon observed in our material differs from A. eunotioides mainly in the valve dimensions, being much smaller, and the higher density of the marginal spines. Stria density also tends to be coarser. The valve length of A. eunotioides var. minor, on the other hand, corresponds with the smallest valves of our taxon but the valves are narrower, with a higher stria density. The ventral margin is slightly convex, while in the new taxon it is slightly concave. Moreover, in the original description of A. eunotioides var. minor, the absence of spines is mentioned, while they are present on the valves in the materials we investigated. Based on the observed morphological differences, we consider the taxon recorded in the streams and small rivers in the Yangambi Biosphere Reserve distinct from A. eunotioides and its var. minor and subsequently, we describe Actinella kufferathiana as a species new to science.

Illuminating the problem of blue verditer synthesis in the early modern English period: chemical characterisation and mechanistic understanding

We present a study into early modern English production of blue verditer, an early synthetic copper-based blue pigment chemically analogous to azurite. Verditers have been identified in numerous wall and easel paintings. While initial documentation occurs in the mid 1500s and production recipes were documented by the 17th c., the synthesis was known to be unreliable. This study replicates historical and recent scientific work on blue verditer and represents a significant advance in our understanding of verditer production and its challenges. Procedures for verditer synthesis are drawn from both 17th c. documentation and 20th c. replication work. The effects of temperature, copper and carbonate sources, solution stirring, copper ion concentration, and atmospheric composition are studied in order to elucidate the mechanism of synthesis and explain its unreliability in early modern refineries. Products are characterised by polarised light microscopy, scanning electron microscopy, Raman spectroscopy, and powder X-Ray diffraction. Rouaite, a green basic copper nitrate, is for the first time confirmed as a product of the refiners’ synthesis and a precursor to blue verditer in laboratory syntheses. This result problematises the blanket identification of green verditer as basic copper carbonate and provides important clues to the mechanism for blue verditer synthesis. Solution chemistry and ion equilibria allow us to explain the route by which rouaite is first formed and then converted to blue verditer. Conditions favouring blue verditer production are also clarified further. Although it is commonly stated that low temperatures are required for blue verditer production, blue verditer is produced here at a range of ambient temperatures. The reaction is found instead to be controlled by solution equilibria and heavily favoured by high partial pressures of carbon dioxide. Alongside archival materials about refining and verditer production, these results are contextualised and explanations for the unreliability of historical synthesis are proposed.

Electromagnetic drop-on-demand (DoD) technology as an innovative platform for amorphous solid dispersion production

Production of amorphous solid dispersions (ASDs) is an effective strategy to promote the solubility and bioavailability of poorly water soluble medicinal substances. In general, ASD is manufactured using a variety of classic and modern techniques, most of which rely on either melting or solvent evaporation. This proof-of-concept study is the first ever to introduce electromagnetic drop-on-demand (DoD) technique as an alternative solvent evaporation-based method for producing ASDs. Herein 3D printing of ASDs for three drug-polymer combinations (efavirenz-Eudragit L100-55, lumefantrine-hydroxypropyl methylcellulose acetate succinate, and favipiravir-polyacrylic acid) was investigated to ascertain the reliability of this technique. Polarized light microscopy, differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and Fourier Transform Infrared (FTIR) spectroscopy results supported the formation of ASDs for the three drugs by means of DoD 3D printing, which significantly increases the equilibrium solubility of efavirenz from 0.03 ± 0.04 µg/ml to 21.18 ± 4.20 µg/ml, and the equilibrium solubility of lumefantrine from 1.26 ± 1.60 µg/ml to 20.21 ± 6.91 µg/ml. Overall, the reported findings show how this new electromagnetic DoD technology can have a potential to become a cutting-edge 3D printing solvent-evaporation technique for on-demand and continuous manufacturing of ASDs for a variety of drugs.

A Pilot Study of Aerosolization of InfectiousMurine Norovirus in an Experimental Setup.

Human norovirus is transmitted mainly via the faecal-oral route, but norovirus disease outbreaks have been reported in which airborne transmission has been suggested as the only explanation. We used murine norovirus (MNV) as a surrogate for human norovirus to determine the aerosolization of infectious norovirus in an experimental setup. A 3-l air chamber system was used for aerosolization of MNV. Virus in solution (6 log10 TCID50/ml) was introduced into the nebulizer for generating aerosols and a RAW 264.7 cell dish without a lid was placed in the air chamber. Cell culture medium samples were taken from the dishes after the aerosol exposure time of 30 or 90min, and the dishes were placed in a 37°C, 5% CO2 incubator and inspected with a light microscope for viral cytopathic effects (CPEs). We determined both the infectious MNV TCID50 titre and used an RT-qPCR assay. During the experiments, virus infectivity remained stable for 30 and 90min in the MNV solution in the nebulizer. Infectious MNV TCID50 values/ml of 2.89 ± 0.29 and 3.20 ± 0.49 log10 were measured in the chamber in RAW 264.7 cell dish media after the 30-min and 90-min exposure, respectively. The MNV RNA loads were 6.20 ± 0.24 and 6.93 ± 1.02 log10 genome copies/ml, respectively. Later, a typical MNV CPE appeared in the aerosol-exposed RAW cell dishes. We demonstrated that MNV was aerosolized and that it remained infectious in the experimental setup used. Further studies required for understanding the behaviour of MNV in aerosols can thus be performed.

Biogenic synthesis of Psidium guajava‐mediated silver nanoparticles: A comprehensive antibiofilm and antivirulence study

AbstractIn this study, at sub‐MIC, the efficacy of green synthesized Psidium guajava‐mediated silver nanoparticles (Pg@AgNPs) in inhibiting quorum sensing facilitated virulence factors and suppressing biofilm formation was evaluated against clinical multidrug resistant (MDR) isolates, specifically Chromobacterium violaceum MCC‐2290 and Pseudomonas aeruginosa PAO1. The AgNPs were synthesized by adding the leaf extract to a solution of silver nitrate, and the reduction of Ag+ ions to Ag0 was confirmed by the appearance of brown color. The Pg@AgNPs were characterized using Fourier transform infrared (FTIR), UV/Vis spectroscopy, TEM, and XRD. The UV/Vis spectrum showed the characteristic absorption peak of Pg@AgNPs in the range of 350–480 nm. FTIR analysis substantiated the existence of functional groups implicated in the synthesis and stabilization of AgNPs. The XRD analysis elucidated the crystalline characteristics of the nanoparticles. The TEM images further confirmed the spherical shape and size of the nanoparticles, which ranged between 5 and 25 nm. The AgNPs demonstrated notable antibacterial efficacy against C. violaceum and P. aeruginosa. Moreover, the Pg@AgNPs effectively suppressed both biofilm formation and the production of quorum sensing‐mediated virulence factors such as pyoverdin (77.2%), pyocyanin (78.12%), and violacein (83.4%). Additionally, the Pg@AgNPs demonstrated broad‐spectrum activity against biofilm formation, showing the biofilm inhibition in C. violaceum and P. aeruginosa by 81.86% and 87.89%, respectively. Under light microscopy, it is also observed that Pg@AgNPs reduce the formation of biofilm in both tested bacteria. The present finding highlights the potential of green‐synthesized AgNPs using P. guajava leaf extract as antimicrobial agents with potential applications in combating bacterial infections and biofilm‐related diseases.

Neurokinin B Administration Induces Dose Dependent Proliferation of Seminal Vesicles in Adult Rats.

Neurokinin B; an endogenous decapeptide, mediates its reproductive physiological actions through gonadotropin releasing hormone. Despite the potential role of Neurokinin B on seminal vesicles, its effects on seminal vesicles in adult male mammals remain elusive. We aimed to investigate the potentials of variable doses of Neurokinin B, its agonist and antagonist on histomorphology and expression of NK3R on seminal vesicles, and secretory activity of seminal vesicles in adult male rats. Adult male Sprague Dawley rats (n=10 in each group) were administered intraperitoneally with Neurokinin B in three variable doses: 1 μg, 1 ηg and 10 ρg while, Senktide (Neurokinin B agonist) and SB222200 (Neurokinin B antagonist) in 1 μg doses consecutively for 12 days. After 12 days of peptide treatment, half of the animals (n=05) in each group were sacrificed while remaining half (n=05) were kept for another 12 days without any treatment to investigate treatment reversal. Seminal vesicles were dissected and excised tissue was processed for light microscopy, immunohistochemistry and estimation of seminal fructose levels. Treatment with Neurokinin B and Senktide significantly increased while SB222200 slightly decrease the seminal vesicles weight, epithelial height and seminal fructose levels as compared to control. Light microscopy revealed increased epithelial height and epithelial folding as compared to control in all Neurokinin B and Senktide treated groups while decreased in SB222200. Effects of various doses of Neurokinin B, Senktide and SB222200 on seminal vesicles weight, epithelial height, seminal fructose levels and histomorphology were reversed when rats were maintained without treatments. Immuno-expression of Neurokinin B shows no change in treatment and reversal groups. Continuous administration of Neurokinin B and Senktide effect positively while SB222200 have detrimental effects on cellular morphology, epithelial height and seminal fructose levels in seminal vesicles. Effects of peptide treatments depicted a reversal towards control group when rats were kept without any treatment.

Clustered Circulating Tumor Cells as a Predictor of Adjuvant-chemotherapy Efficacy in Lung Cancer

BackgroundSurrogate markers of minimal residual disease primarily include cell-free tumor DNA and circulating tumor cells. Cell-free tumor DNA might aid precise decision-making regarding who should receive adjuvant chemotherapy. However, there are no relevant reports on circulating tumor cells. Therefore, we aimed to verify whether perioperative clustered circulating tumour cells identification is a predictor of therapeutic efficacy in non-small cell lung cancer adjuvant chemotherapy. MethodsCirculating tumor cells were diagnosed under light microscopy using a size selection method in 128 patients with clinical stage I/II non-small cell lung cancer around surgery. The main endpoint was recurrence-free survival, and the effect of adjuvant chemotherapy was verified in both groups based on perioperative clustered circulating tumor cell identification. ResultsIn total, 49 and 79 patients were included in the clustered circulating tumor cell-positive and clustered circulating tumor cell-negative patient groups, respectively. In the clustered circulating tumor cell-positive patient group, adjuvant chemotherapy was performed in 18 patients (2-year recurrence-free survival rate, 71.8%). However, the 2-year recurrence-free survival rate was 36.3% in 31 patients who did not receive adjuvant chemotherapy (P < .01). In the clustered circulating tumor cell-negative patient group, adjuvant chemotherapy was provided in 11 patients (2-year recurrence-free survival rate, 90.9%). However, 68 patients did not receive adjuvant chemotherapy (2-year recurrence-free survival rate, 94.9%) (not significant). ConclusionsIn surgical cases of clinical stage I/II non-small cell lung cancer, patients with perioperative clustered circulating tumor cells had a poor prognosis, but adjuvant chemotherapy improved their prognosis.

Actin cytoskeletal proteins in mice cortical microvessels decreased with aging

Background: The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and differentiation. The interactions of actin microfilaments with plaque proteins zonula occludens (ZO)-1, -2, -3 are required ensuring the formation of a polarized brain endothelium which ultimately drives development of blood-brain barrier (BBB). In this study, we carried out an extensive evaluation of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery-based quantitative proteomic approach. Methods: Cortical MVs were isolated from equal number of age-matched, male and female, young (4–6 months), middle-aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1-EGFP)MJrs/J] and bred in a C57B16J background (n = 6/group). The presence of end-arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry. Results: We quantified 4746, 4216, and 4579 differentially expressed proteins by proteomics in brain cortical MVs of young, middle-aged, and old mice, respectively. We found that several actin cytoskeletal-related alpha actinin-1, -2, -3 (ACTN1/2/4), actin-related protein -2, -3, -3B (ACTR2/3/3B), actin-related protein 2/3 complex subunit -1A, -2, -4, -5 (ARPC1A/2/4/5), and [F-actin]-monooxygenase MICAL3 proteins were significantly downregulated in mice cortical MVs with aging. Under physiological conditions, cerebral endothelial cells are connected by tight junctions (TJs) and adherens junctions (AJs). The TJ and AJ proteins are anchored to the actin cytoskeleton by multiple accessory proteins, ZO-1, ZO-2 and ZO-3. Interestingly, we observed that several TJ, AJ, and their signaling (PKC-α/γ/ε, RHOA, RAB3 and YES1) proteins were significantly downregulated in mice cortical MVs with aging. Moreover, we previously documented that oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability and ATP synthesis capacity in mice. Conclusions: We suggest that increased oxidative stress during aging leads to protein expression profile changes of brain cortical MVs that affect ATP synthesis capacity, which leads to actin cytoskeletal dysregulation, and accelerating to endothelial and BBB dysfunction in the brain microvasculature with aging. Funding: AG075988, HL148836, AG063345, AG074489, NS114286, AG047296, HL141143, HL168568, and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Feasibility study on hybrid manufacturing – combining laser‐based powder‐bed fusion and chill casting on the example of EN AC‐42000 alloy

AbstractLaser‐based powder‐bed fusion can be combined with casting into a hybrid manufacturing process. This is done to simultaneously utilize advantages of both processes. To examine related challenges stemming from this combination when using aluminium alloys, first experiments were conducted with EN AC‐42000 alloy, an alloy common in both standalone processes. EN AC‐42000 alloy samples with different surface treatments were placed into a sand mould and cast on to with an aluminium melt. The contact region between the partially molten insert and the cast material was examined with light microscopy. The main challenges were shown to be comprised of bonding issues due to present oxides and a high porosity. The high porosity was traced back to a porosity increase in the laser‐based powder‐bed fusion insert.

Heterogeneity of mesenchymal cells in human amniotic membrane at term.

There is increasing interest in understanding the tissue biology of human amniotic membrane (hAM) given its applications in medicine. One cellular component is mesenchymal cells, which can be extracted, cultured and differentiated "in vitro" into various cell types. These studies show that there is heterogeneity among mesenchymal cells. The aim of this work is to study the membrane in situ to determine whether this cellular heterogeneity exists. The hAMs were obtained from caesarean deliveries at term and analyzed by histological techniques. Types I-III mesenchymal cells and Hofbauer were distinguished by light microscopy. Histochemically, mesenchymal cell types showed successively increasing positivity to: PAS, vimentin, fibronectin, and Concanavalin-A; VGEF, TGF-β2, PDGF-C, FGF-2. By the semiquantitative point of view, the percentage of Type II cells was 60%, significantly higher than the other types. With transmission electron microscopy, an intermediate cell type between II-III was observed. Strong vesiculation of the rough endoplasmic reticulum (RER) with exocytosis was observed. In addition, an accumulation of a similar material to the extracellular matrix in the RER caused its dilation especially in type IIITEM cells. Some of this material acquired a globular structure. These structures were also found free in the extracellular matrix. In conclusion, the mesenchymal cells of the fibroblastic layer of the hAMs studied are heterogeneous, with some undifferentiated and others with a probably senescent fibroblastic phenotype with accumulation in their RER of fibronectin. These results may be of interest to extract mesenchymal cells from hAMs for use in regenerative medicine and to better understand the mechanisms of fetal membrane rupture.

Detecting SARS-CoV-2 in bats of New Mexico using immunohistochemistry on formalin- fixed, paraffn-embedded tissues

Objectives: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and its zoonotic origin have raised critical questions regarding the potential role of local bat populations as reservoirs or intermediates for the transmission of this or related coronaviruses to humans. The goal of this project is to investigate the presence and diversity of coronaviruses in bat species native to the Southwest USA. Broadly, this study has two primary aims: (1) to detect antigens of coronaviruses in tissues of local bat populations via histological methods and (2) to perform a molecular screen of coronaviruses in these bats. Methods: Individuals from five bat species were evaluated: Artibeus jamaicensis, Myotis velifer, Antrozous pallidus, Tadarida brasiliensis, and Eptesicus fuscus. All species were local, wild-caught bats, with the exception of Artibeus jamaicensis, which is from a colony established at New Mexico State University (NMSU). Tissue was sourced from bats collected by TJO at NMSU. Bat tissue was fixed with formalin and samples subsequently taken of lung, kidney, nasal epithelium, and uterus. Tissue was further processed and embedded in paraffn wax. The embedded tissue was sectioned via a rotary microtome at 4 μm thickness and mounted onto slides. Slides were stained with general coronavirus anti-nucleocapsid primary antibody and a hematoxylin counterstain to visualize cellular structures. Slides were cover-slipped with DPX mounting medium and observed via light microscopy. Results: The immunohistochemistry protocol was performed on tissue from each of the five bat species investigated and on human placenta samples known to be positive for SARS-CoV-2. The latter was used as positive controls. Putative positive staining was seen in both human placenta and bat tissues. Interestingly, coronavirus was detected in Artibeus jamaicensis samples, an unexpected finding given its status as a laboratory colony. Verification of this putative finding is ongoing. Further work on our samples will determine the genetic identity of the coronaviruses detected and contribute to developing a molecular screen for more effcient detection of coronavirus antigens in local bat tissue. This work will serve as a springboard for further understanding coronavirus ecology and evolution in bats of the US southwest, and it can be used to further inform both local surveillance efforts and public policy surrounding the interaction between humans and animals in the natural environment and in controlled settings. Conclusions: Putative detection of coronavirus antigens was found in tissues from bat samples from the Southwestern USA. Implications for this work include broadening our understanding of the diversity and abundance of coronaviruses in the US. Burrell College; US National Science Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hydralazine use can be associated with IgM-dominated immune complex-mediated glomerulonephritis

ABSTRACT Context IgM-dominant immune complex-mediated glomerulonephritis (IgM-dominant ICMGN) is a rare renal entity, characterized by a membranoproliferative pattern by light microscopy, dominant IgM staining by immunofluorescent staining, and subendothelial deposits by electron microscopy. This study was to investigate if some of IgM-ICMGN were associated with autoimmune disorders induced by hydralazine. Design Seven IgM-dominant ICMGN cases were identified over 8 years. Their pathologic phenotypes and clinical scenarios were analyzed in detail. Results Patients’ ages ranged from 47 to 87 years old with 5 women and two men. Six of seven patients had drug-induced autoimmune phenomenon (hydralazine-induced positive ANCA and ANA). All of them had renal dysfunction and some proteinuria. Most pathologic features showed a membranoproliferative pattern of glomerulonephritis with dominant IgM deposits at subendothelial spaces. IgM nephropathy (a variant of focal segmental glomerulosclerosis), chronic thrombotic microangiopathy, and cryoglobulinemic glomerulopathy were ruled out in the cases. Conclusion The hydralazine-induced autoimmune phenomenon can be seen in IgM-dominant ICMGN, which should be classified as a subtype of membranoproliferative glomerulonephritis.

Defining Architectural Changes in Mouse Pelvic Floor Muscles During Pregnancy to Withstand Physiological Distension Associated with Parturition

Pelvic floor disorders (PFDs), which are present in ~25% of women, profoundly impact quality of life. During childbirth, pelvic floor muscles (PFMs) endure substantial mechanical strain, which can lead to PFM dysfunction. As such, vaginal childbirth is a major risk factor for PFDs. Previous studies in rats demonstrate important structural adaptations in the PFMs that help to withstand the mechanical strain of childbirth, including muscle fiber lengthening by sarcomerogenesis. However, it is diffcult to establish a comprehensive understanding of the molecular mechanisms underlying PFM dysfunction during childbirth in rat models. Here, our objective was to determine if mouse PFMs undergo similar adaptations to rats during pregnancy and vaginal distension. We hypothesized that, akin to rats, PFMs in mice would undergo muscle fiber elongation through sarcomerogenesis during pregnancy. The PFMs (coccygeus [C], iliocaudalis [ICa], pubocaudalis [PCa]) and a non-pelvic floor muscle (tibialis anterior [TA]) were collected from late-pregnant (E16.5) and non-pregnant C57BL/6NJ (3 months old) mice. These animals were subjected or not subjected to physiological vaginal distension to mimic vaginal parturition (N = 4/group); in anesthetized mice, a 6F transurethral catheter was inserted into the vagina, and it was inflated with 0.3 mL (which best approximated fetal head circumference), and a 13 g weight was attached to the catheter creating circumferential and downward strains similar to parturition. Following this, the animals were euthanized, and the pelvis was fixed in situ for assessment of muscle architectural parameters; muscle fibers length was determined using digital calipers and sarcomere length was measured by light microscopy. Pregnancy induced a significant increase in normalized muscle fiber length in all PFMs compared to non-pregnant animals (Pregnant vs. not pregnant — C: 4.71±0.06 mm vs. 3.37±0.15 mm, p&lt;0.0001; ICa 8.74±0.14 mm vs. 7.50±0.27 mm, p=0.004; PCa 7.13±0.09 mm vs. 6.28±0.34 mm, p=0.04); TA muscle fiber length was unchanged (6.92±0.17 mm vs. 6.73 ± 0.19 mm, p=0.28). Sarcomere length was not affected by pregnancy in any muscle (C: 2.44±0.002 μm vs. 2.45 ± 0.003 μm, p=0.55; ICa: 2.26±0.003 μm vs. 2.26±0.004 μm, p=0.64; PCa: 2.16±0.01 μm vs. 2.16±0.003 μm, p=0.74; TA: 2.55±0.004 μm vs. 2.54±0.003 μm, p=0.28). Balloon-mediated vaginal distention resulted in significantly longer sarcomere lengths in non-pregnant compared to pregnant animals in all PFMs (C: 2.98±0.01 μm vs. 2.43±0.01 μm, p&lt;0.0001; ICa: 2.54±0.004 μm vs. 2.24±0.005 μm, p&lt;0.0001; PCa: 2.85±0.001 μm vs. 2.17±0.001 μm, p&lt;0.0001), indicating protection against sarcomere elongation in pelvic floor muscles of pregnant animals. This study shows that, similar to rats, pregnancy causes architectural changes in mouse pelvic floor muscles, which prevent excessive sarcomere stretching during physiologic vaginal distension that is comparable to fetal delivery. This work was funded by NIH grant #K12 HD000849 (Reproductive Scientist Development Program) to LAB and UC San Diego Senate Grant to LAB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physics-based modeling of the effective gas transport properties of single jersey knitted fabrics based on images

Physics-based modeling of the effective gas transport properties of single jersey knitted fabrics based on images

Podocytopathy and Nephrotic Syndrome in Smpdl3b Gene Knockout Mice

Recent clinical studies have shown that downregulation of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in podocytes is a hallmark of idiopathic nephrotic syndrome in children. Meanwhile, there is increasing evidence suggesting that changes of SMPDL3b level in podocytes determine the phenotypes of podocyte injury in glomerular diseases. However, it remains unknown whether SMPDL3b exclusively plays a crucial role in the pathogenesis of podocytopathy and nephrotic syndrome without any other pathogenic stimuli. In the present study, we tested whether Smpdl3b gene knockout induces podocyte injury and nephrotic syndrome in mice. By immunohistochemistry and confocal microscopy, we first confirmed that SMPDL3b was highly expressed in podocytes of wild type (WT) mice but undetectable in these cells of Smpdl3b knockout (KO) mice. Also, Western blot analysis showed that SMPDL3b was not detected in podocytes isolated from Smpdl3b KO mice compared to podocytes of WT mice. Although no significant morphologic changes in glomeruli were observed in Smpdl3b KO mice under light microscope, severe proteinuria and albuminuria were found in Smpdl3b KO mice compared to control littermates. Transmission electron microscopy showed that podocytes of Smpdl3b KO mice had distinctive foot process effacement and moderate microvillus formation and vacuolation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice lacking Smpdl3b gene. In vitro, Smpdl3b gene silencing induced remarkable decrease in podocin levels in podocytes compared to control cells. Phalloidin staining of F-actin showed elevation of cortical actin and reduction of stress fiber in podocytes transfected with Smpdl3b shRNA. Such actin cytoskeleton rearrangement induced by Smpdl3b gene silencing was significantly attenuated by choline but exaggerated by CDP-choline, which are product and substrate of SMPDL3b, respectively. Furthermore, atom force microscopy was used to study mechanical properties of podocytes lacking Smpdl3b gene as the outcome of actin cytoskeleton reorganization. Smpdl3b gene deletion was found to increase podocyte height but decrease cortical elasticity in these cells. Moreover, these pathological changes were markedly inhibited by choline. Taken together, our findings suggest that SMPDL3b is essential for the maintenance of functional and structural integrity of podocytes and that Smpdl3b gene knockout may induce podocytopathy and nephrotic syndrome in mice. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.