Glass Slides Research Articles

PSIII-11 Impacts of late gestational in utero heat stress on small intestine dynamics of neonatal dairy calves

Abstract Late gestation heat stress in dairy cows has been shown to negatively affect offspring growth and development. In utero heat stressed calves have altered small intestine morphology and an impaired ability to absorb colostral immunoglobulin G (IgG). Thus, the objective of this study was to characterize potential cellular and molecular alterations in the small intestine of neonatal in utero heat stressed calves. In the last ~56 d of gestation pregnant dams were either heat stressed (shade; n = 41) or cooled (shade, fans, soakers; n = 41) resulting in heifers that were in utero heat stressed (IUHS, n = 15) or in utero cooled (IUCL, n = 15). Heifers were fed 3.78 L of high-quality colostrum within 2 h of birth. Apparent efficiency of absorption (AEA) of IgG was calculated by measuring IgG content fed in colostrum and in 24 h serum. Within 4.6 ± 2.3 h of birth a subset of calves (n = 8/treatment) not fed colostrum were euthanized to collect samples from the ileum. Tissue for histological analysis was placed in 10% neutral buffered formalin before being dehydrated, embedded in paraffin, cut into 5 μm thicknesses, and mounted to glass slides. The TUNEL method was used to identify apoptotic cells using the Keyence BZ-X800 Microscope. Tissue for gene expression analysis was snap-frozen and kept at -80°C before RNA extraction and cDNA synthesis. The transcript abundance of endosomal (FCGRT, DAB2, MAMDC4), tight junction (ZO1, ZO2, ZO3, OCLN, CLDN1), and heat shock (HSP90, HSF1) related genes were measured by qPCR using the CFX96 Real-Time PCR detection System (BIO-RAD). Data were analyzed using PROC MIXED or PROC GLIMMIX depending on the response variable. TUNEL results are expressed as the proportion of positive cells to total cells. Quantitative PCR data were analyzed by assessing the estimates of the model relative to the geometric mean of 3 reference genes (ACTB, GADPH, RSP9; ΔΔCT). The concentration of colostrum IgG fed to calves was not different (136.1 vs. 122.5 g/L; IUHS vs. IUCL, respectively; P = 0.32), but AEA of IgG was reduced in IUHS relative to IUCL calves (23.5 vs. 33.3%; P < 0.01). The proportion of apoptotic cells within the villi of the ileum was increased in IUHS calves compared with IUCL (2.11 vs. 1.57%; P < 0.01). Ileum gene transcript abundance of DAB2 and MAMDC4 were increased (P < 0.01), while FCGRT transcripts tended to increase in IUHS calves (P = 0.08). All tight junction and heat shock-related gene transcripts were increased within the ileum of IUHS calves (P ≤ 0.02) with the exceptions of CLDN1 and HSP90, which were not different between treatments (P ≥ 0.22).These results suggest that exposure to heat stress in utero impact small intestine integrity after birth and affect key IgG transporters, providing insight into underlying mechanisms responsible for reduced IgG absorption in IUHS calves.

A beginner’s guide to immunohistochemistry

Using the affinity between antibodies and antigens, immunohistochemistry (IHC) is a helpful method for analysing the cellular components within tissue sections placed on glass slides. The chemistry of antibodies was not discovered until 1941, and since then, their use has increased due to the discovery of enzymes and fluorochromes conjugated to antibodies, enabling observations through optical or fluorescence microscopy (FM). Hence, immunofluorescence (IF), or IHC with fluorescent antibodies, has been associated with the advancement of FM and fluorochromes. Specifically, the discovery of three fluorochromes that emit blue, red and green led to a rise in IF application in the context of visualizing numerous components in a single cell. The proximity ligation assay (PLA) method, so termed because the antibody is conjugated with short DNA strands called PLA probes, is the most recent development in the field of antibody conjugation. Protein–protein interactions and sub-localization of proteins can both be studied using this technique. The improvement of conjugation techniques has occurred in parallel with the development of sophisticated microscopies, including confocal and slide scanners. Confocal microscopy allows for the reconstruction of three-dimensional structures while a slide scanner can digitally convert up to 100 slides' worth of high-resolution pictures. The first step in the IHC process is the collection and fixation of tissues from animal models and humans. Paraformaldehyde is the most commonly used fixative. Fixation prevents antigen blocking and creates cross-links between proteins to preserve tissue structures. Fixation is followed by cutting tissues using microtome or cryostat, then slides are treated for antigen exposition (antigen retrieval) and then included in Triton X solution, which increases membrane permeability. Subsequently, incubations with enzyme- or fluorochrome-conjugated antibodies enable observations via optical microscopy or FM, respectively. In clinical practice and research, IHC and IF are still helpful methods for identifying the antigen-proteins linked to specific diseases.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-04.

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.

Abstract PO5-26-09: Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67

Abstract Background and Aims Ki-67 is a well-established biomarker of proliferation in breast cancer (BC). However, its value in treatment decision making is hampered by a lack of analytical reproducibility. Regular participation in external quality assessment (EQA) substantially improves inter-laboratory concordance. Pre-requisites in establishing a fit-for-purpose EQA for Ki-67 are a well-validated testing substrate and a reproducible method of assessing Ki-67 scores in participants’ submitted materials. We report here on the results of work using cell line controls analysed by digital image analysis (DIA) as a first step towards providing such an EQA. Materials and Methods A formalin-fixed paraffin embedded (FFPE) cell line microarray (CLMA) was designed and produced in conjunction with Array Sciences LLC (Sausalito, USA). It was comprised of cores taken from a pure population of Sf9 caterpillar cells, which have been shown to be completely unreactive with most commercial antibodies to human Ki-67, together with cores of Sf9 cells mixed with four different human BC cell lines. These were BT-20 (85% BC cells), ZR-75-1 (75%), BT-474 (65%) and BT-483 (55%). Sections from the CLMA were mounted onto glass microscope slides together with sections from a FFPE tonsil sample and two BC samples; one BC showed high (~30%, BC-high) and the second, low proliferation (~5%, BC-low). In both BC samples proliferating cancer cells were distributed homogeneously throughout the the block. Unstained sections were distributed to laboratories participating in the Scheme’s Ki-67 BC programme; after routine IHC-staining for Ki-67 returned slides were centrally visually assessed for stain quality and subjected to DIA using an in-house application developed on Visiopharm software (Visiopharm A/S, Hoersholm, Denmark). Results Slides were returned by 37 laboratories. Analysis of Ki-67 scores obtained on the Sf9 core identified two distinct groups. The first (n = 27) were negative or showed low Ki-67 scores (mean = 1.1%, 95% CIs: 0.2-1.9%), the second (n = 10) displayed a step-change in scores (mean = 49.9%, 95% CIs: 33.2-66.7%); the means of the two groups were significantly different (P< 0.0001). When Ki-67 scores for each of the tissue samples were dichotomized into similar groups, the means of those groups differed significantly for the two BC samples (P< 0.001), but not for tonsil. Quality scores generated by visual assessment did not differ significantly between the two groups. However, when slides bearing Sf9 cores demonstrating aberrant Ki-67 scores were visually examined nuclear staining was clearly visible, and non-specific nuclear staining could also be identified in the matched BC tissue samples, but not in the tonsil sections. Correlation of Ki-67 scores between the four BC cell line cores and each of the BC tissue samples was examined using Pearson’s correlation statistics. The r statistic range was 0.57-0.71 in comparisons between BC cell line cores and the BC-high sample, and 0.53-0.70 for those with BC-low; in each case BT-483 showed the highest correlation score and BT-20 the lowest. A similar analysis was undertaken between the tonsil and the two BC tissues. The r statistic for correlation between tonsil and BC-high scores was 0.72; it was 0.64 for those between tonsil and BC-low. Conclusions By using a pure population of Sf9 cells we have developed a sensitive indicator of non-specific nuclear staining in IHC preparations stained for Ki-67 which identifies the presence of the artefact quantifiably. Cores made from Sf9/BC cell line mixtures (especially BT-483) produce Ki-67 scores which correlate with those obtained in breast cancer samples at a similar level to those achieved between tonsil and BC samples; this is true for both high and the low proliferation ranges. Cell line mixtures can be adjusted to show Ki-67 scores in the clinically relevant ranges, and they do not show the inherent biological variations seen in tissue controls such as tonsil. Citation Format: Andrew Dodson, Fitim Berisha, Dawn Wilkingson, Lila Zabaglo, Suzanne Parry. Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-09.

Abstract PO2-13-06: Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response

Abstract Background: Therapeutic cancer vaccines aim to stimulate the immune system by utilizing tumor antigens to trigger an antitumor response. In our clinical trials, we have focused on evaluating the efficacy of the breast cancer cell line secreting GM-CSF, SV-BR-1-GM as a therapeutic vaccine, and we have observed encouraging clinical outcomes. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 – study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 – study ongoing). To further improve the therapeutic efficacy of this vaccine we have initiated a study to characterize patient’s immune response to this treatment. It has been demonstrated that cancer vaccines can generate both humoral and cellular immune responses. However, predicting immune responses to cancer vaccines, especially when whole cells are employed as immunogens, presents significant challenges. We present here a preliminary analysis of the antibody response to SV-BR-1-GM in breast cancer patients using antigen arrays. Methods: Large-scale protein arrays are versatile and sensitive platforms for antibody specificity evaluation. The HuProt™ Human Proteome Microarray (CDI Laboratories, Inc., Baltimore, Maryland, United States) provides the largest number of unique, full-length, individually purified human proteins on a single microscope slide. This allows thousands of interactions to be profiled in a high-throughput manner. We utilized here an unbiased human protein microarray platform encompassing >21,000 proteins and isoforms from ~19,000 unique genes to identify IgG and IgM responses against self-antigens elicited by treatment. Serum samples from SV-BR-GM-treated patients were analyzed, comparing pre- and post-treatment. The human serum samples were probed at 1:1000 dilution. After sample processing and data collection the raw signal intensities on all arrays were quantile normalized using CDI software. Heatmaps were generated using MetaboAnalyst 5.0, which utilizes the R pheatmap package (version 0.7.7). For paired analysis, fold changes (FCs) were calculated by determining the ratio between paired pre- and post-treatment samples, resulting in one FC per pair. The means of these FCs (pair means) were then computed. ANOVA tests were performed using the RStatix R package, which automatically determines the appropriate Type I, II, or III errors for the analysis. Results: By using this approach, we were able to evaluate a broader range of antigens compared to previous investigations. Using a variety of statistical approaches, potential correlations with patient survival on SV-BR-1-GM were evaluated. Antibody responses to galectin antigens demonstrated the most consistent relationships with survival. Further confirmation with additional patients and prospective analysis are needed to fully understand relationships with clinical benefit and survival. Citation Format: Miguel Lopez-Lago, Mingjin Chang, Giuseppe Del Priore, Vikas Bhardwaj, Patience Cournoo, Pedro Ramos, Tyler Hulett, Charles Wiseman, William Williams. Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-06.

Oil blotting paper for formalin fixation increases endoscopic ultrasound-guided tissue acquisition-collected sample volumes on glass slides.

Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is used for pathological diagnosis and obtaining samples for molecular testing, facilitating the initiation of targeted therapies in patients with pancreatic cancer. However, samples obtained via EUS-TA are often insufficient, requiring more efforts to improve sampling adequacy for molecular testing. Therefore, this study investigated the use of oil blotting paper for formalin fixation of samples obtained via EUS-TA. This prospective study enrolled 42 patients who underwent EUS-TA for pancreatic cancer between September 2020 and February 2022 at the Osaka International Cancer Institute. After a portion of each sample obtained via EUS-TA was separated for routine histological evaluation, the residual samples were divided into filter paper and oil blotting paper groups for analysis. Accordingly, filter paper and oil blotting paper were used for the formalin fixation process. The total tissue, nuclear, and cytoplasm areas of each sample were quantitatively evaluated using virtual slides, and the specimen volume and histological diagnosis of each sample were evaluated by an expert pathologist. All cases were cytologically diagnosed as adenocarcinoma. The area ratios of the total tissue, nuclear, and cytoplasmic portions were significantly larger in the oil blotting paper group than in the filter paper group. The frequency of cases with large amount of tumor cells was significantly higher in the oil blotting paper group (33.3%) than in the filter paper group (11.9%) (p = 0.035). Oil blotting paper can increase the sample volume obtained via EUS-TA on glass slides and improve sampling adequacy for molecular testing.

Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Comparative Study of Modified Ultrafast Papanicolaou and Standard Papanicolaou Staining Technique for the Assessment of Cervical Smears

Introduction: Cervical invasive carcinoma is one of the most frequent cancers in the world, and it can be prevented with screening. The papanicolaou (Pap) smear is the most common screening procedure. Pap staining, as it is now done, is costly, requires a large amount of alcohol, and takes a long time. Materials and Methods: A total of 170 patients were recruited. One hundred and sixty-six patients, considering eligibility criteria, undergoing Pap smear test. Eligibility criteria: Age: 18–49 years females, who did not have a total hysterectomy done, who consented to participate in the study. Each patient had two smears taken on glass slides. Standard Pap smears were fixed for at least 15 min in 95% ethanol. The other smear was air-dried before being fixed with alcohol formalin and stained with modified ultrafast Pap (MUFP) stain. A comparison of two different techniques was made on the basis of cytomorphological features. Results: In studied smears, a clean background was seen in 18.24% and 14.12% of the cases in Pap stain and MUFP, respectively. Kappa analysis found a good correlation between Pap stain and MUFP (kappa: 0.81, P < 0.01). Crisp chromatin was seen in 89.41% and 67.65% of MUFP and Pap staining, respectively. About 88.82% of MUFP stains showed optimal cytoplasmic details and the same was revealed by 81.18% of Pap stains. MUFP has a better quality index (QI) as compared to Pap staining as no case in MUFP had QI of < 0.80 with a statistically significant difference as P < 0.05. Conclusion: MUFP is a simple, user-friendly, affordable, and less time-consuming alternative in low-resource areas in comparison to the traditional technique for mass cervical cancer screening.

In High Glucose Exposed Endothelial Cells BLNK and BTK are Novel Components of the Nuclear Factor-kappa B Pathway

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFκB signaling in ischemic endothelial cells (EC). We have previously shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFκB pathway through activation of protein kinase C β (PKCβ). However, the signaling pathways upstream of PKCβ involved in this high glucose induced desensitization of NFκB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFκB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates from NG, HG, NGI and HGI groups were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spots by the signal intensity of the corresponding non-phosphorylated spots for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling by associating with the scaffold protein BLNK. However, neither of these proteins has been known to be expressed in ECs or play a role in EC function. In this study, we confirmed BLNK and BTK protein and transcript expression in ECs. Inhibition of BTK by a selective inhibitor terreic acid decreased PKCβ-S661 phosphorylation and restored NFκB activation through IκBα in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as potentially new components of the NFκB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. National Heart, Lung, and Blood Institute (R01 HL130399) to AOD. 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.

Prevalence of Salmonella spp., Shigella spp., and intestinal parasites among food handlers working in University of Gondar student's cafeteria, Northwest Ethiopia.

Food-borne infections continue to be a major public health problem at the international level. The issue becomes more serious in developing countries like Ethiopia. This study aimed to examine the prevalence of Salmonella and Shigella species and intestinal parasites, as well as antimicrobial resistance patterns and associated factors among food handlers at the University of Gondar cafeteria in northwest Ethiopia. An institutional-based cross-sectional study was conducted from February to June 2021 in the University of Gondar cafeterias. Data related to the socio-demographic characteristics and hygienic practices of study participants were collected using structured questionnaires. A total of 290 stool samples were collected from food handlers. Culture and conventional biochemical tests were used to isolate the Salmonella and the Shigella species. Wet mount, Formol-ether concentration, and Kato Katz techniques were applied to identify intestinal parasites. Additionally, drug susceptibility tests were performed using the disk diffusion method. Statistical analysis was done using SPSS version 26. Of 290 food handlers' stool samples analyzed, Twenty-seven 27 (9.3%) were positive for both Salmonella and Shigella species. The prevalence of Salmonella and Shigella species was 16 (5.5%) and 11 (3.8%), respectively. Most of the isolated pathogens were resistant to tetracycline 19 (70.4%), and trimethoprim/sulphamethoxazole 19 (70.4%). The overall rate of multi-drug resistant Shigella and Salmonella isolate was 59.3%. Besides, Fifty-seven 57 (19.7%) of the participants were positive for one or more intestinal parasites. The most prevalent intestinal Parasitosis was E. histolytica/dispar 22 (7.6%), followed by G. lamblia 13 (4.5%), and Ascaris lumbricoides 11 (3.8) not washing hands after using the toilet (AOR: 4.42, 95% CI: 1.57, 10.56), and consuming unpasteurized milk (AOR: 3.14, 95% CI: 1.65, 3.96), were factors significantly associated with the prevalence of Salmonella, and Shigella infection. Similarly, not washing hands after using the toilet (AOR: 2.19, 95% CI: 1.0, 1.4), and consuming unpasteurized milk (AOR: 10.4, 95% CI: 3.8, 28.8), were factors significantly associated with the prevalence of intestinal parasites infection. The prevalence of intestinal parasites, Salmonella, and Shigella species was high. Therefore, it is imperative to implement a public health policy that includes ongoing microbiological surveillance.

Performance prediction of a novel solar power tower system based on spectral beam splitting

Photovoltaic technology is currently the most widely used solar generation technology in engineering. The particular spectral response band of PV cells cannot convert all solar radiation into electricity. Meanwhile, some radiation will become heat, leading to an increase in cell temperature and a decrease in the conversion efficiency of photovoltaic cells. Spectral splitting technology is an effective technology to improve electrical generation efficiency and can achieve full spectrum utilization of solar energy. The current work involved (1) proposing a novel solar power tower comprehensive utilization system based on spectral splitting; (2) developing a spectral splitting thin film configuration by using the Needle method whose transmittance is higher than 90% against the band range of 510 nm-1, 020 nm; (3) setting up a spectral splitting PV panel whose cover glass is coated the developed film, and replacing the heliostat of SPT system by using such spectral splitting PV panels; (4) predicting the electrical power generation of the spectral splitting-based tower type concentrating solar power system. The results demonstrate that the power generation efficiency of the proposed system is 5.5% and 3.46% higher than PV power generation and SPT systems respectively.

Structured illumination with infrared imaging for measuring thermal conductivity

With developments in advanced manufacturing and materials by design comes the need for high-throughput thermal characterization and inspection. Towards this end, Structured Illumination with Thermal Imaging (SITI) is an all-optical pump-probe thermal characterization technique recently developed by our group. In the first generation [Zheng et al., Appl. Phys. Rev. 9, 021411 (2022)] SITI uses an LED with a digital micromirror device (DMD) to “structurally illuminate” and heat the sample with dynamic patterns, a visible light camera for thermoreflectance based “thermal imaging” [leveraging a Microsanj MTIR120], and the resultant temperature response was fit with a thermal model to characterize the sample’s thermal properties. This represents a novel approach to dynamic and flexible spatial mapping of thermal properties by virtue of being a non-contact technique and having a simpler scanning means (computer control only) than conventional pump-probe laser methods. SITI also can tolerate rough samples with diffuse reflections. This talk presents the second generation of SITI. The pumping is now based on a lower cost off-the-shelf digital projector. The thermometry is now performed using an infrared (IR) camera, which we find is a more flexible and accessible hardware approach compared to the thermoreflectance microscopy used previously. With these updates the setup can deliver higher heating power and a broader range of frequencies, allowing an extended range of samples that can be studied. We have demonstrated SITI’s ability to measure the thermal conductivity of a microscope glass slide.

Population dynamics of the brown planthopper (Nilaparvata lugens Stảl.) in rice fields and their association with the entomopthoralean fungus

The rice plant (Oryza sativa L.) is one of the primary food sources for Indonesian people. The Brown planthopper (BPH), Nilaparvata lugens is one of the most devastating rice insect pests in Indonesia. The entomopthoralean fungus, Pandora delphacis is reported to be able to infect BPH naturally in the rice field. This research aims to identify the entomopthoralean fungi phase and determine the level of fungal infection and its relations with the population dynamic of BPH in rice fields. This research was conducted in 2015 and 2016 on rice fields at Bogor Regency and Subang Regency; and in 2017 at Karawang Regency, West Java. The sampled BPH were collected from rice fields. The microscope slide squash mounts in lactophenol cotton blue were made for the insects and it was examined with a microscope compound to determine if hyphal bodies, conidiophore, primary conidia, secondary conidia, saprophytic fungi, and resting spores were present. The results showed that the entomopthoralean fungi phase found on BPH are hyphal bodies, primary conidia, secondary conidia, and saprophytic fungi. The fungus infected all the phases of the BPH, from the first nymphal phase to adult.

Performance analysis of a novel solar Linear Fresnel concentrator system based on spectral beam splitting

Photothermal and photovoltaic are the most common and commercialized ways of utilizing solar energy. However, they cannot use solar energy efficiently. SBS (spectral beam splitting) technology is an efficient solar energy utilization technology that can realize the full spectrum utilization of solar energy. The current work involved: (1) Proposing a SBS-LFCs (linear Fresnel concentrator system) based on spectral beam splitting. In this system, the SBS photovoltaic panel replaces the conventional linear Fresnel concentrator lens, which can realize photovoltaic and photothermal utilization at the same time; (2) Developing a spectral splitting thin films configuration using the Needle method whose transmittance is higher than 90% against 380 nm-1100 nm band range; (3) Designing the SBS photovoltaic panel structure. The cover glass surface is covered with a SBS thin film. The SBS photovoltaic panel replaces the concentrator lens of LFCs; (4) Taking four typical days as examples, the annual power generation of a SBS linear Fresnel focusing system was predicted. The outcome indicates that the total conversion efficiency of solar thermal power in the system is 23.8%, 23.5%, 23.5%, and 23.6% on the spring equinox, summer solstice, autumn equinox, and winter solstice, respectively. Compared to conventional monocrystalline silicon cells, the photoelectric conversion efficiency has been improved by 14.6%, 12.8%, 12.5%, and 14.1%, respectively.

High-efficiency fabrication of uniform photonic crystals by a vertical deposition method from multiple glass slides of simple moulds and their application in isomer recognitions

Vertical deposition method is a simple, cheap and common method for fabricating photonic crystals (PCs). However, a majority of PCs are obtained based on the traditional vertical deposition method from one glass slide, the method is low efficiency. Herein, it is proposed to efficiently fabricate uniform PCs by an optimized vertical deposition method from double or multiple glass slides of simple moulds. Amounts of SiO2 spheres, distances of two glass slides of moulds, preparation temperatures and different diameters of SiO2 spheres are investigated for obtaining uniform PCs. And different types of PCs (the bottom-middle-top 3D PCs, bilayer 2D PCs and amorphous PCs) are also fabricated based on the optimized vertical deposition method. The above method is a simple, cheap and high-efficiency way for mass production of uniform PCs. Meanwhile, the chemicals with similar refractive indices (isomers and cis-trans isomers) can be easily distinguished under high humidity based on dynamic reflection spectra of the PCs. For example, the difference in the refractive indices of m-xylene and p-xylene is only 0.002, the difference of desorption balance time of PCs is 263 s. The PCs have potential applications in visual detections, isomer recognitions and monitoring of volatilization rates of trace liquids.

Recent advancement in the synthesis of zinc sulphide thin films

This research work has given an overview on the preparation of zinc sulphide thin films on different types of substrates (quartz, soda lime glass, silicon, polyethylene terephthalate film, fluorine-doped tin oxide glass, indium tin oxide coated glass, silica, stainless steel, mica, microscopy glass slide and (100) GaAs substrate) using different types of deposition methods. XRD data showed information about the different phases (cubic, wurtzite and hexagonal) presented in the obtained as-deposited films and annealed samples. Based on optical studies, the band gap was observed in the range of 2.6 eV to 3.87 eV with good absorption coefficient value (105 to 106 cm-1). Experimental results indicated these films could be used in solar cell applications, anti-reflection coating, photocatalysts, photo detector devices and laser-oriented coating applications.

Design modification of elliptical vessel solar receiver by response surface methodology

Design modification of elliptical vessel solar receiver system by response surface methodology has been carried out. The materials used in this study were locally sourced from Kenyeta Market Enugu, Onitsha Bridge Head Market, and Idumota Market Lagos. These materials were sourced based on categories of components element: support mechanisms made of mild steel plates, bolts, nuts, clamps, and water as heat transfer fluid. The reflector is made of aluminum foil tape while the vessel has a glass cover fitted with bolts and nuts, the receiver is made of copper pipe, aluminum pipe, galvanized iron pipes, and stainless steel pipes. The pipes were fitted into the vessel with chlorinated polyvinyl chloride 3⁄4 joint pipes, and journal-bearing mechanisms. Other features include the tracking system made of light dependent resistance sensors, a direct current motor, a pulley, a belt, an Arduino controller, and a thermal energy storage tank. The lagging material was an expanded Polyethylene sheet. Experimental data were measured with thermocouples, a digital panel, a Uni-T digital anemometer (UT363), and a digital solar power meter (SM206-SOLAR). Matrix Experimental design was used to develop an experimental model for the system. The developed system was tested to investigate the effect of various heat collectors with and without coating on its performance. It was established from the response optimization that the intercept factor was improved by 32.2%. Similarly, the theoretical efficiency was improved by 8.19% while the experimental thermal efficiency was improved by 6.99%.

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase.

Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

Optimization of crystal violet technique for enhanced fingerprint detection on various surfaces.

The crystal violet (CV) staining technique represents a prevalent approach for the development of latent fingerprints, especially on adhesive tape surfaces. Nevertheless, the technique necessitates intricate formulations to augment its performance. In this investigation, an optimized CV staining protocol was developed, characterized by the absence of residual dye on the target substrates and the capability of facilitating fingerprint visualization under ambient light conditions. Four donors, comprising two males and two females, deposited natural fingerprints on various substrates, including glass microscope slides, aluminum foil, and 115 g glossy coated paper, without any specific guidelines. Fingerprints developed using cyanoacrylate fuming served as benchmarks and were contrasted with those generated through alternative methods: CV, ardrox, rhodamine 6G, powdering, and the optimized CV staining protocol. The fingerprint development experiment was replicated at seven distinct time intervals, encompassing 1 day, 1 week, 1, 3, 6, 9, and 12 months, resulting in a dataset of 420 fingerprints. The evaluation of fingerprint identifiability employed a scoring system established by the Home Office Centre for Applied Science and Technology. The results indicated that the optimized CV staining technique demonstrated superior performance, boasting a 92.9% rate of identifiable fingerprint development in contrast to other employed methodologies. Consequently, this optimized CV staining approach is recommended as an efficient, rapid, and straightforward critical dyeing method, applicable to a wide array of substrates in forensic investigations.