Light Microscopy Research Articles

B-351 Genotoxic Producing Escherichia coli Protects and Promotes Breast Adenocarcinoma

Abstract Background Recent studies have linked certain colibactin producing subphylas of E. coli to colorectal cancer. The bacterium synthesizes colibactin via the pks genomic island containing a series of clbA to clbS genes. The genotoxin causes inter-strand DNA cross-links, which lead to double strand breaks and promotion of colorectal adenocarcinoma. The published studies have prioritized the colorectal regions of the body where E. coli is commonly found, but none have identified the same genotoxic relationship with other types of adenocarcinoma. In this study, we evaluate DNA damage and cellular changes of mammary breast tissue based on high and low levels of colibactin producing E. coli exposure. The goal is to evaluate if colibactin can affect cells in the mammary breast tissue promoting breast adenocarcinoma growth. Methods Two cell lines were acquired from ATCC: primary mammary epithelial cells (PCS-600-010) and MCF-7 breast adenocarcinoma cells (HTB-22). Both cell lines were split into six culture flasks and maintained until ∼80% confluence was reached using the ATCC cell culture protocols. Once all the flasks had reached the target confluence, they were divided into exposure groups of high and low concentrations of colibactin producing E. coli, a group of non-colibactin producing E. coli, a group exposed only to phosphate buffered saline, and a control group with no exposure. Cellular morphology was observed using light microscopy before, after exposure, and every 5 minutes up to 15 minutes. E. coli and colibactin DNA concentrations were quantified using quantitative polymerase chain reaction. Genomic DNA was extracted from each group of cells. The DNA from each group was whole genome sequenced and analyzed for genomic variation. Results The normal cells exposed to E. coli showed immediate lysing and continued to lyse after a 5 minute incubation; however, the adenocarcinoma positive cells showed no lysing up to 30 minutes incubation. Sequencing results show genomic variation between the groups and specific break points associated with the E. coli exposed groups. Conclusions Our results show that the presence of E. coli with mammary breast tissue can destroy normal tissue, promote lysis resistance, and allow adenocarcinoma cells to continue to grow. The colibactin producing E. coli does cause double stranded breaks and can lead to cell death. This new information points out a critical need to monitor the microbiome and infection levels of people at risk or in early stages of cancer to prevent adenocarcinoma growth and destruction of normal tissue. This study indicates that colibactin producing E. coli may play a larger role in tissue damage and promotion of other types of cancer. More research is needed to elucidate the molecular pathway and mechanisms of colibactin, which will lead to new diagnostics or therapeutics that monitor E. coli levels or inhibit colibactin’s toxic effects.

Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections.

Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)-Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host-parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae.

Hypoxia and Normoxia Preconditioned Olfactory Stem Cells Against Noise-Induced Hearing Loss.

Noise-induced hearing loss is one of the leading causes of permanent hearing loss in the adult population. In this experimental study, the authors aimed to investigate the effectiveness of hypoxia and normoxia preconditioned olfactory stem cells against noise trauma. Twenty-seven female guinea pigs were enrolled. Two guinea pigs were sacrificed for harvesting olfactory tissue and 1 for examining the architecture of the normal cochlea. The remaining 24 guinea pigs were exposed to noise trauma for 1 day and then randomly divided into 3 groups: intracochlear injection of (i) normoxic olfactory stem cells, (ii) hypoxic olfactory stem cells, and (iii) physiological serum. Auditory brainstem response (ABR) measurement was performed before and 2 weeks after noise trauma and weekly for 3 weeks following intracochlear injection. Both click and 16kHz tone-burst stimuli were used for detection of ABR. No significant difference was noted between the groups before and 2 weeks after noise trauma. ABR thresholds detected after intracochlear injections were significantly higher in the control group compared with stem cell groups. However, no significant difference was detected between the stem cell groups. Fluourescence microscopy revealed better engraftment for hypoxic stem cells. Light and electron microscopy examinations were consistent with predominant degenerative findings in the control group, whereas normoxic group had more similar findings with normal cochlea compared with hypoxic group. Olfactory stem cells were demonstrated to have the potential to have beneficial effects on noise trauma.

Novel adomavirus associated with proliferative skin lesions affecting the dermal denticles of a sand tiger shark (Carcharias taurus).

A captive sand tiger shark (Carcharias taurus) presented with progressive, hard, raised, miliary skin lesions localized to the lateral trunk and peduncle. Histopathologic evaluation of biopsy samples revealed dysplastic proliferation of odontogenic epithelium with the production of collagenous material. Inclusion bodies and viral particles were not observed with light or transmission electron microscopy, respectively. However, using next generation sequencing with Illumina MiSeq and PCR followed by Sanger sequencing, the complete genome of a novel adomavirus, tentatively named sand tiger shark adomavirus (STAdoV), was obtained from the affected tissue. The genome was circular and 18.5 kilobases with bidirectionally transcribed genes, namely EO1, EO2 & 4, EO3, LO4, LO5, LO6, LO7, LO8, and SET. In situ hybridization using RNAscope® technology and a STAdoV specific probe localized viral DNA to the nuclei of proliferating epithelial cells. Adomaviruses are an emerging viral group with structural and replicative genes sharing a complex evolutionary history with adenoviruses and small circular DNA tumor viruses including papillomaviruses and polyomaviruses. Adomaviruses are described in a number of fish species in association with both necrotizing and proliferative diseases. BLAST analysis of the viral genome revealed greatest nucleotide identity (71.29%) to guitarfish adomavirus (GAdoV), another elasmobranch virus associated with proliferative (epidermal) skin lesions. Lesions in the index animal persisted for approximately 1 year during which time four conspecifics developed similar proliferations. Ultimately, lesions in all sharks regressed spontaneously without recurrence for 2 years.

Microstructure evolution after hot working and heat treatment in 6082 aluminum alloy manufactured by horizontal continuous casting

In this paper, 6082 aluminum alloy manufactured by horizontal continuous casting was subjected to high-temperature plastic deformation under 20–70 % deformation levels with following T6 heat treatment process. Microstructural and phase changes were investigated by light and scanning electron microscopy together with energy dispersive spectroscopy, while grain, grain boundary, dislocation and texture evolution was studied by electron-backscattered diffraction. The results are showing that continuous dynamic recrystallization processes are active during the hot working while influence of static recrystallization is significant only for highest deformation degrees. The abnormally coarse-grained microstructure does locally occur in the subsurface layer after the 70 % deformation and T6 heat treatment. Crystallographic texture components are gradually evolving from the various types (Goss, Brass, D) for the low deformations (up to 40 %) into fiber-type textures (α-fiber, γ-fiber or C2-fiber) for high deformations due to the inhomogeneity of plastic deformation. The formation of AlMnSi dispersoids, CSL-boundaries Σ25b and an increase in fraction of GND, MgSi and Fe-based intermetallic particles was observed during the phase transformation which promotes ongoing nucleation of recrystallization processes.

Morphological structure of salivary glands, alimentary canal, and Malpighian tubules in adult Eurydema spectabilis Horváth, 1882 (Heteroptera, Pentatomidae).

Eurydema spectabilis (Heteroptera: Pentatomidae) has a piercing-sucking mouth type and feeds on plant sap. In this study, the morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis was examined using light and scanning electron microscopy. Salivary glands consist of the principal and accessory salivary glands. In E. spectabilis, digestion begins in the mouth and ends in the anus. Alimentary canal is divided into three parts: foregut, midgut, and hindgut. The foregut consists of pharynx, esophagus, and proventriculus. The esophagus connects to the proventriculus and resembles a narrow tube. The wall of the proventriculus has a recessed structure and is surrounded by a single cylindrical layer of epithelium and muscle. The midgut is divided into three regions: the first, second, and third ventricles (V1-V3). V1 and V2 consist of single-layered cylindrical epithelium. V3 contains a single layer of cuboidal epithelium. Gastric caeca were found in the midgut. The hindgut consisted of a pylorus followed by a well-developed rectum. The wall of the rectum consists of a single-layer cuboidal epithelium and muscle. Its lumen contains numerous bacteria and uric acid crystals. The pylorus consists of a single-layered cylindrical epithelium. It is also the origin of Malpighian tubules. Malpighian tubules consist of two regions: proximal and distal. The morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis, which has not been studied before, was examined and discussed in comparison with other orders. It is also aimed to contribute to future studies. RESEARCH HIGHLIGHTS: In E. spectabilis, the salivary glands are divided into principal and accessory salivary glands. Microvilli and numerous secretory granules were found in Malpighian tubules. Numerous uric acid crystals and bacteria were found in the rectum lumen.

Mitochondrial Structure, Dynamics, and Physiology: Light Microscopy to Disentangle the Network.

Mitochondria serve as energetic and signaling hubs of the cell: This function results from the complex interplay between their structure, function, dynamics, interactions, and molecular organization. The ability to observe and quantify these properties often represents the puzzle piece critical for deciphering the mechanisms behind mitochondrial function and dysfunction. Fluorescence microscopy addresses this critical need and has become increasingly powerful with the advent of superresolution methods and context-sensitive fluorescent probes. In this review, we delve into advanced light microscopy methods and analyses for studying mitochondrial ultrastructure, dynamics, and physiology, and highlight notable discoveries they enabled.

Metformin in combination with chemotherapy increases apoptosis in gastric cancer cells and counteracts senescence induced by chemotherapy.

Gastric cancer (GC) is the fourth leading cause of cancer death in the world, and there is a demand for new therapeutic agents to treat GC. Metformin has been demonstrated to be an antineoplastic agent in some types of cancer; however, it has not been sufficiently valued in treating GC because the effect of metformin in combination with chemotherapy regimens has not yet been evaluated. The present study aimed to evaluate the mechanisms underlying cell death induced by metformin alone or when combined with chemotherapy. The cytogenetic characteristics of the NCI-N87 cell line were determined by fluorescence in situ hybridization (FISH). To determine viability, the cells were treated with metformin, epirubicin, cisplatin, docetaxel and 5-fluorouracil (individually and at different concentrations). Subsequently, the cells were treated with metformin alone, and in combination with the chemotherapeutic drugs and the epirubicin + cisplatin + 5-fluorouracil, docetaxel + cisplatin + 5-fluorouracil, and cisplatin + 5-fluorouracil regimens. Cell viability, proliferation and mitochondrial membrane potential (ΔΨm) were analyzed by spectrophotometry. Apoptosis, caspase activity and cell cycle progression were assessed by flow cytometry. Finally, light microscopy was used to evaluate senescence and clonogenicity. The results revealed that metformin, alone and when combined with chemotherapy, increased the proportion of apoptotic cells, promoted the loss of ΔΨm, and induced apoptosis through caspase activity in GC cells. Moreover, metformin decreased cell proliferation. In addition, metformin alone did not induce senescence and it counteracted the effects of chemotherapy-induced senescence in GC cells. Additionally, metformin, alone and when combined with chemotherapy, decreased the clonogenic capacity of NCI-N87 GC cells. In conclusion, metformin may increase the effects of chemotherapy on NCI-N87 cell death and could represent an option to improve the treatment of GC.

Severity of Kidney Dysfunction in Obstructing Tubulopathy Is Associated with the Number of Renal Tubular Obstructing Foci by Light Microscopy

Severity of Kidney Dysfunction in Obstructing Tubulopathy Is Associated with the Number of Renal Tubular Obstructing Foci by Light Microscopy

‘A comparative study of traditional and molecular diagnostic methods for detection of gastrointestinal parasites in Nepalese migrants to the UK’

BackgroundWe evaluated the results of examining a single faecal sample for gastrointestinal parasites (GIP) using a combination of traditional methods with multiplex qPCR for helminths and protozoa, compared to a reference standard of examining three faecal samples from each person using traditional diagnostic methods alone. MethodsThree faecal samples were collected at weekly intervals from 596 healthy Nepalese men. Each sample underwent formalin-ethyl acetate (FEA) concentration and light microscopy, and charcoal culture. The combined results of these investigations for all three stool samples were designated the reference standard. The first sample was also analysed using a multiplex TaqManTM qPCR assay, screening for five helminths and three protozoa. We compared sensitivity and specificity of analysing the first faecal sample with qPCR alone, or a hybrid approach combining qPCR with traditional methods, to the reference standard. Additionally, a serum sample was taken from each participant for Strongyloides stercoralis IgG ELISA. ResultsThe reference standard identified 139 GIP infections in 133 (22.3%) participants. Use of qPCR alone in one stool identified 176 infections in 147 (24.8%) participants, rising to 187 infections in 156 (26.3%) when combined with FEA microscopy and charcoal culture. The sensitivity of this latter hybrid approach was 100% for Strongyloides spp., 90.9% for Trichuris trichiura, 86.8% for hookworm species and 75% for Giardia duodenalis compared to the reference standard. The hybrid approach increased the detected cases of G. duodenalis by 4.5% (46 cases) overall, T. trichiura by 2.9% (18 cases), Strongyloides spp. by 1% (6 cases), and hookworm by 0.5% (8 cases), compared to the reference standard. ConclusionExamination of a single faecal sample using qPCR alone showed superior or equivalent sensitivity to traditional methods for most GIP infections when both were compared to the reference standard. Combining molecular and traditional methods to analyse a single stool improved the detection rate for most studied parasites. This approach has value in settings where repeated sampling and/or faecal culture for helminths is impractical, but molecular diagnostics are available.

Combined interventions for the testing and treatment of HIV and schistosomiasis among fishermen in Malawi: a three-arm, cluster-randomised trial

Undiagnosed HIV and schistosomiasis are highly prevalent among fishermen in the African Great Lakes region. We aimed to evaluate the efficacy of lakeside interventions integrating services for HIV and male genital schistosomiasis on the prevalence of schistosomiasis, uptake of antiretroviral therapy (ART) for HIV, and voluntary male medical circumcision (VMMC) among fishermen in Malawi. We conducted a three-arm, cluster-randomised trial in 45 lakeshore fishing communities (clusters) in Mangochi, Malawi. Clusters were defined geographically by their home community as the place where fishermen leave their boats (ie, a landing site). Eligible participants were male fishermen (aged ≥18 years) who resided in a cluster. Clusters were randomly allocated (1:1:1) through computer-generated random numbers to either enhanced standard of care (SOC), which offered invitation with information leaflets to a beach clinic offering HIV testing and referral, and presumptive treatment for schistosomiasis with praziquantel; peer education (PE), in which a nominated fisherman was responsible for explaining the study leaflet to promote services to his boat crew; or peer distribution education (PDE), in which the peer educator explained the leaflet and distributed HIV self-test kits to his boat crew. The beach clinic team and fishermen were not masked to intervention allocation; however, investigators were masked until the final analysis. Coprimary composite outcomes were the proportion of participants who had at least one Schistosoma haematobium egg observed on light microscopy from 10 mL of urine filtrate and the proportion who had self-reported initiating ART or scheduling VMMC by day 28. Outcomes were analysed by intention to treat; multiple imputation for missing outcomes was done; random-effect binomial models adjusting for baseline imbalance and clustering were used to compute unadjusted and adjusted risk differences, risk ratios (RRs) and 95% CIs, and intracluster correlation coefficients for each outcome. This trial is registered with ISRCTN, ISRCTN14354324. Between March 1, 2022, and Jan 29, 2023, 45 (65·2%) of 69 clusters assessed for eligibility were enrolled in the trial, with 15 clusters per arm. Of the 6036 fishermen screened at baseline, 5207 (86·3%) were eligible for participation: 1745 (87·6%) of 1991 in the enhanced SOC group, 1687 (81·9%) of 2061 in the PE group, and 1775 (89·5%) of 1984 in the PDE group. Compared with the prevalence of active schistosomiasis in the enhanced SOC group (292 [16·7%] of 1745), 241 (13·6%) of 1775 fishermen in the PDE group (adjusted RR 0·80 [95% CI 0·69-0·94]; p=0·0054) and 263 (15·6%) of 1687 fishermen in the PE group (0·92 [0·79-1·07]; p=0·28) had schistosomiasis at day 28. 230 (13·2%) in the enhanced SOC group, 281 (16·7%) in the PE group, and 215 (12·1%) in the PDE group initiated ART or were scheduled for VMMC. ART initiation or VMMC scheduling was not significantly increased with the PDE intervention (0·88 [0·74-1·05); p=0·15) and was marginally increased with the PE intervention (1·16 [0·99-1·37]; p=0·069) when compared with the enhanced SOC group. No serious adverse events were reported in this trial. We found weak evidence for the use of peer education to increase uptake of ART and VMMC, but strong evidence for the added distribution of HIV self-test kits to promote high engagement with services and reduce the prevalence of active schistosomiasis, suggesting a high potential for scale-up in hard-to-reach communities across Malawi. Wellcome Trust and the UK National Institute for Health Research.

Scanning of antennae and maxillary palps of anthropophilic Aedes aegypti and ornithophilic Culex pipiens as potential arbovirus vectors

Background and Aim: Efficient mosquito vectors are required to persist and propagate arthropod-borne diseases that seriously affect impoverished populations worldwide. Mosquito sensilla plays a crucial role in host-seeking and disease transmission to humans. This study aimed to distinguish between the several types of sensilla found on the antennae and maxillary palps of Culex pipiens and Aedes aegypti, matching this diversity with host preference and disease transmission. Materials and Methods: Overall, 1300 mosquitoes were collected and examined using dissection and light microscopy. Scanning electron microscopy was used to identify and describe the diverse types of sensilla found on the antennae and maxillary palps of C. pipiens and A. aegypti. Results: In total, 900 C. pipiens and 400 A. aegypti mosquitoes were identified. The antennae and maxillary palps of C. pipiens and A. aegypti carry both sensilla trichoidea and sensilla chaetica. The C. pipiens antenna has long and short grooved peg sensilla, whereas A. aegypti lacks long pegs and expresses only occasional short pegs. The maxillary palps express Capitate pegs in both mosquito species and exclusively show sensilla campaniform in A. aegypti. Conclusion: The lack of long-grooved pegs and the presence of few short pegs, along with campaniform sensilla, limit the host range of A. aegypti and reduce its susceptibility to many infections, unlike C. pipiens. Keywords: Aedes aegypti, Culex pipiens, Scanning electron microscopy, Sensilla.

Molecular Typing of Cryptosporidium in Cattle in Federal Capital Territory, Nigeria

Cryptosporidium is an enteric pathogen with worldwide distribution in humans and animals with cattle as their reservoirs. Molecular tools have been very useful in determining the epidemiology of this zoonotic pathogen of public health importance. Despite the importance of this pathogen to humans and animals, there is limited published information on the risk factors of its epidemiology in the Federal Capital Territory of Nigeria. Positive faecal samples of 400 cattle in 46 farms located in 6 area councils of the FCT that were earlier screened for Cryptosporidium on light microscopy were genotyped by nested PCR using primers that targeted 18S ssUrRNA gene and followed by RFLP. The PCR-RFLP products were further sub genotyped by nested PCR and GP 60 KDa gene amplification. The nucleotides of the amplicons were subsequently sequenced. The overall PCR detection rate was 5.5%. Analysis of the 18S rRNA gene of the PCR-RFLP fragments revealed Cryptosporidium ryanae (18.3%), C. andersoni (8.5%) and C. parvum (4.2%). Analysis of the nucleotide sequence of the GP 60KDa gene of all the C. parvum detected showed all to be 100% subtype family IIa and 100% sub genotype IIaA18G3R1. In conclusion, this study has established the presence of Cryptosporidium in cattle in the FCT. The detection of the zoonotic C. parvum, its subtype IIa and its allele IIaA18G3R1 is an indication of source of zoonotic transmission of the parasite in the FCT, Nigeria.

Association between body mass index and urinary tract infections: A cross-sectional investigation of the PERSIAN Guilan cohort study.

There is a relationship between excess body weight and the risk of a number of infectious diseases, including urinary tract infections (UTIs). This study aimed to investigate the correlation between body mass index (BMI) and UTIs among Prospective Epidemiological Research Studies of the Iranian Adults (PERSIAN) Guilan Cohort Study (PGCS) population. This cross-sectional study was conducted on 10,520 individuals aged 35-70 years from PGCS. The demographical data and clinical characteristics of the participants were recorded. Microscopic examination of the urine samples was performed to detect the presence of bacteria or white blood cells (WBC) as indicators of infection. UTI was defined as the presence of bacteria in the urine (Few, moderate, and many) and a value of ≥10 WBC/high power field (HPF) by light microscopy. The prevalence of UTIs in this study was 8.8%, with a higher incidence in females compared to males (12.2% vs. 4.7%, p<0.001). Among participants, the prevalence of UTIs across different weight categories was as follows: underweight/normal weight, 7.1%; overweight, 8.1%; and obesity, 10.9%. According to the unadjusted model, subjects with obesity were at significantly higher odds for UTIs than subjects with underweight/normal BMI (OR=1.62, 95% CI: 1.35-1.93, p<0.001). However, this association was no longer significant after adjusting for demographic and clinical variables. The findings of this study provide evidence supporting a higher prevalence of UTIs among individuals with obesity.

In vitro leishmanicidal activity of Hancornia speciosa latex against Leishmania (Leishmania) amazonensis

BackgroundLeishmaniasis is a neglected tropical disease whose available treatment has limitations. Hancornia speciosa Gomes, a tree native to Brazil, has been studied for its medicinal properties, including antimicrobial and healing properties. However, its effect on parasites of the Leishmania genus still needs to be elucidated. PurposeTo evaluate the leishmanicidal activity for potential therapeutic use of H. speciosa latex and its fractions on Leishmania (Leishmania) amazonensis promastigotes and amastigotes, as well as to analyze its cytotoxic effects on RAW264.7 cells and murine peritoneal macrophages. MethodsThe anti-promastigote activity of latex and its fractions was evaluated by cell viability assays via MTT in L. (L.) amazonensis and monitoring the parasite's growth kinetics by counting under light microscopy. To evaluate anti-amastigote activity, RAW264.7 cells and murine peritoneal macrophages infected with amastigotes were analyzed using microscopy, immunofluorescence, and flow cytometry assays. The cytotoxicity of latex and its fractions was evaluated in these cells via MTT assay. ResultsThe H. speciosa latex and its fractions showed anti-promastigote activities in L. (L.) amazonensis, reducing its proliferation and viability and altering its morphology. These did not interfere with the viability of RAW264.7 cells and peritoneal macrophages. Crude latex and F<10 kDa at 5% reduced the infection of intracellular amastigotes in peritoneal macrophages to 32% and 18% after 24 h of treatment, respectively. Under the same conditions, the F>10 kDa fraction showed 82% of infected cells. The evaluation of the latex composition showed that the F>10 kDa fraction has the highest concentration of carbohydrates, followed by phenolic compounds and proteins. In contrast, the F<10k Da fraction presented fewer phenolic compounds and carbohydrates. ConclusionsThese results indicate that H. speciosa crude latex and F<10 kDa have intense leishmanicidal activity against L. (L.) amazonensis promastigotes and amastigotes internalized by macrophages. Additionally, the lack of cytotoxicity indicates that H. speciosa latex is a strong candidate for new alternative treatments for leishmaniasis.

KMnO4/Pb staining allows uranium free imaging of tissue architectures in low vacuum scanning electron microscopy

Scanning electron microscopy under low-vacuum conditions allows high-resolution imaging of complex cell/tissue architectures in nonconductive specimens. However, the conventional methods for metal staining of biological specimens require harmful uranium compounds, which hampers the applications of electron microscopy. Here, we introduce a uranium-free KMnO4/Pb metal staining protocol that allows multiscale imaging of extensive cell/tissue architectures to intensive subcellular ultrastructures. The obtained image contrast was equivalent to that of Ur/Pb staining and sufficient for ultrastructural observation, showing the fine processes of podocytes in the glomerulus, which were invisible by light microscopy. The stainability in the elastic tissue indicated that the distinct histochemical properties of KMnO4 oxidation led to Pb deposition and BSE signal enhancement superior to Ur staining. Elemental analysis clarified that the determinant of the backscattered electron signal intensity was the amount of Pb deposition enhanced by KMnO4 oxidation. This user-friendly method is anticipated to create a new approach for biomedical electron microscopy.

Concordance of whole slide imaging and conventional light microscopy for assessment of pathologic response following neoadjuvant therapy for lung cancer

Pathologic response is an endpoint in many ongoing clinical trials for neoadjuvant regimens, including immune checkpoint blockade and chemotherapy. Whole slide scanning of glass slides generates high resolution digital images and allows for remote review and potential measurement with image analysis tools, but concordance of pathologic response assessment on digital scans compared to glass slides has yet to be evaluated. Such a validation goes beyond previous concordance studies which focused on establishing surgical pathology diagnoses, as it requires quantitative assessment of tumor, necrosis, and regression. Further, as pathologic response assessment is being used as an endpoint, such concordance studies have regulatory implications. The purpose of this study was two fold: firstly, to determine the concordance between pathologic response assessed on glass slides and on digital scans; and secondly, to determine if pathologists benefited from using measurement tools when determining pathologic response. To that end, H&E-stained glass slides from 64 non-small cell lung carcinoma specimens were visually assessed for percent residual viable tumor (%RVT). The sensitivity and specificity for digital vs. glass reads of complete pathologic response (pCR, 0% RVT) and major pathologic response (MPR, ≤10% RVT) were all >95%. When %RVT was considered as a continuous variable, intraclass correlation coefficient of digital vs. glass reads was 0.94. The visual assessments of pathologic response were supported by pathologist annotations of residual tumor and tumor bed areas. In a separate subset of H&E-stained glass slides, several measurement approaches to quantifying %RVT were performed. Pathologist estimates strongly reflected measured %RVT. This study demonstrates the high level of concordance between glass slides evaluated using light microscopy and digital whole slide images for pathologic response assessments. Pathologists did not require measurement tools to generate robust %RVT values from slide annotations. These findings have broad implications for improving clinical workflows and multisite clinical trials.

Atrial septal pouch in cats: first report on interatrial septum anatomical variations.

The atrial septal pouch was first recognised in humans in 2006 and, since 2010, has been described in more detail. The first reports on the left-sided atrial septal pouch linked its occurrence with a higher risk of thromboembolic stroke resulting from left atrial thrombus formation. However, subsequent studies have challenged this theory; therefore, the consequences of septal pouch occurrence remain disputed. Currently, in veterinary species, septal pouch occurrence and morphology have been described in sheep and pigs, showing species-related differences. No studies have been conducted on cats. As cats show a species-related risk of aortic thromboembolic disease resulting from left atrial thrombus formation, we aimed to describe the interatrial septum anatomical variations as the first step in the investigation of the relationship between the septal pouch presence and left atrial thrombus formation. The study was conducted on hearts collected post mortem from 80 cats. After formalin fixation and dissection, a detailed anatomical description of the interatrial septum for all cases was followed by a histopathological analysis of the heart in 25 cases. After dissection, the specimens were embedded in paraffin blocks and stained with haematoxylin and eosin, Masson-Goldner trichrome, Picrosirius Red and Movat pentachrome in a routine manner and evaluated via light microscopy. Our study showed a very high prevalence (95%) of the left-sided septal pouch in cats with a lower incidence (29%) of the right-sided septal pouch. We noted a high morphological variety of the left-sided septal pouch and described anatomical variants not previously reported in the literature. The septal pouch is a common anatomical variation of the interatrial septum in cats. Although, based on the current study, we cannot draw further conclusions on the relationship between the left-sided septal pouch and thromboembolic events in cats, the high prevalence of this anatomical entity points to a need of further studies on the topic.

Spatial Transcriptomic Profiling of Glomeruli in FSGS and Minimal Change Disease That Appear Normal on Light Microscopy

Spatial Transcriptomic Profiling of Glomeruli in FSGS and Minimal Change Disease That Appear Normal on Light Microscopy

Biological effectiveness of combined exposure to neutrons and gamma radiation applied in two orders of sequence: Relevance for biological dosimetry after nuclear emergencies

ObjectiveAfter a nuclear detonation, people will be exposed to varying mixtures of neutrons and gamma radiation and the biological effectiveness of mixed beams is not well known. Additionally, it is not known how far both radiation types interact, a question that is relevant for generating calibration curves for the purpose of biological dosimetry. The objective of this study was to investigate the potential impact of two different combinations of neutron and gamma radiation on gene expression and dicentric chromosomes in peripheral blood mononuclear cells (PBMC). MethodsWhole blood from 3 human donors was exposed to neutrons with an energy spectrum similar to that of the Hiroshima uranium bomb, to gamma radiation from a 60Co source and to a 50:50 combination of both radiations, given in two orders of sequence. In all cases the total doses were 0.5, 0.75 and 1.0 Gy. Dicentric chromosomes were analyzed by light microscopy and the expression of six known radiation-responsive genes BBC3, CDKN1A, FDXR, GADD45A, MDM2, and XPC were analyzed by RT-qPCR. ResultsPer unit dose, exposure to neutrons lead to a higher level of dicentrics and gene expression as compared to gamma radiation. Dose-response relationships for both endpoints were linear, allowing calculating the expected outcome of combined exposure by arithmetic. For dicentric chromosomes, the RBE values for 60Co→neutrons, neutrons→60Co and neutrons were 4.05, 3.62 and 7.30 respectively. For gene expression the RBE values were gene-specific, but showed values in the range of 1.14–3.01 for 60Co→neutrons, 1.33–2.68 for neutrons→60Co and 1.39–3.91 for neutrons. ConclusionsThe results demonstrate that combined exposure to neutrons and gamma radiation, regardless of the order of sequence, leads to an additive response at both endpoints. This indicates that calibration curves for mixed beams can be constructed from dose response relationships of the single beam components.