Staining Of Granules Research Articles

Confused Images Confused Eyes: A Case of Ultrasound Misdiagnosis of Pelvic Actinomycosis.

This article introduces a case of pelvic actinomycosis, which is easily confused with an ovarian malignant tumor. These images are from a 52-year-old woman who was admitted to hospital with difficulty defecating. Colonoscopy and biopsy indicated inflammatory changes within the intestinal tract, but the anti-inflammatory treatment was not effective. Later, she was readmitted due to abdominal pain and emaciation, and laboratory findings revealed mild anemia and inflammation. Various tumor markers are normal. CT suggested inflammatory lesions in the sigmoid colon and upper rectum. PET-CT considered a high metabolic mass originating from the mesentery. Ultrasound scan revealed a mixed-echo mass adjacent to the right side of the uterus, poorly demarcated from the rectum and right ovary, suggesting a neoplastic lesion. A biopsy of the right ovarian mass indicated suppurative inflammation, with negative antacid staining and microscopic observation of yellowish sulfur granules, suggestive of Actinomyces infection. Following a 12-month treatment regimen involving the removal of an intrauterine device and administration of penicillin, the patient's condition markedly improved. Pelvic actinomycosis is usually characterized by abdominal pain accompanied by an abdominal mass, which is often related to an intrauterine device (IUD), and is very difficult to distinguish from pelvic tumors and tuberculosis, so it is necessary for doctors to understand its clinical and imaging features.

Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

Fluorescein staining of chloroplast starch granules in living plants.

Chloroplast starch granules (cpSGs) store energy harvested through photosynthesis in plants, and cpSG dynamics have important roles in plant energy metabolism and stress responses. To date, cpSGs have been visualized using several methods, such as iodine staining; however, no method can be used to specifically visualize cpSGs in living cells from various plant species. Here, we report a simple method to visualize cpSGs in living plant cells in various species by staining with fluorescein, a commonly used fluorescent dye. We show that fluorescein is taken up into chloroplasts and interacts with cpSGs similarly to iodine. Fluorescein also interacts with refined starch in vitro. Using a fluorescein derivative for ultrabright cpSG imaging, we produced high-quality 3D reconstructions of cpSGs and evaluated their accumulation in multiple plant species. As fluorescein is well known and readily purchasable, our fluorescein-based staining method should contribute to all research regarding starch.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. In vitroresults showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium.In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p= 0.046), corneal fluorescein staining (p= 0.021), and International oGVHD Consensus Group severity scores (p= 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.

348-OR: Differentiation of Adult Islet Cell Precursors into ß- and α-like Cells for Islet Transplantation

Advances in protocols to convert human embryonic and inducible pluripotent stem cells (hESCs and iPSCs) into pancreatic β cells has allowed us to generate islet cell organoids in culture for potential use in islet cell transplantation. Here, we demonstrate that dedifferentiated β cells and/or NGN-/RGS16+ islet cell precursors (ICPs) can be harvested from adult pancreatic tissue and converted into β- and α-like cells. In this study, we used ICPs derived from COBE bag pancreatic tissue remnants from organ donors to produce islet cell organoids capable of releasing both insulin and glucagon in response to high (16.7 mM) and low (2.8 mM) glucose conditions, respectively. These organoids displayed zinc-containing granule staining by dithizone and exhibited GSIS with stimulation indices comparable to human islets. ICPs produced genetic profiles similar to hESC-derived endocrine progenitor cells at stages 5-7 (S5-7). Using small molecule ISX9 and GSK/DYRK inhibitors GNF and harmine to induce and activate calcineurin (CN)/NFAT signaling, we were able to expand and differentiate ICPs into β- and α-like cells. CN/NFAT upregulated genes that promote cell cycle and proliferation (S5) and islet cell differentiation genes (S5-7). Islet cell type fate and function could be modulated by hedgehog/GLI signaling. Whereas introduction of sonic hedgehog (Shh) inhibitor SANT-1 at S5 favored production of β-like cells, Shh signaling agonists promoted differentiation to α-like cells up to S7. Overall, these results indicate that adult ICPs can be harvested from donor tissue and expanded for differentiation into functional β- and α-like cells. The capability to expand and differentiate adult ICPs could potentially provide an unlimited supply of islet organoid tissue for islet cell transplantation. Disclosure C.Darden: None. J.Kuncha: None. J.D.Mattke: None. B.Naziruddin: None. M.C.Lawrence: None. Funding Baylor Scott & White Health

"Glycogen deposition in the detrusor muscle of patients with bladder outlet obstruction (BOO) due to benign prostate hyperplasia (BPH); correlation with the urodynamic parameters.''.

A prospective case-control study was conducted to evaluate glycogen deposition within the detrusor and its correlation with the urodynamic findings in patients with bladder outlet obstruction (BOO) due to benign prostate hyperplasia (BPH). Data from 50 patients with BPH (Study Group) and 20 controls (Control Group) were analyzed. BOO was confirmed by pressure-flow studies. The main outcome was glycogen deposition within the bladder wall. Bladder tissue biopsies were obtained from all patients, and histological assessment of the detrusor glycogen content was performed using Periodate Acid Schiff's (PAS) stain. The obtained glycogen score ranged from 0 (no staining of glycogen granules) to 3 (staining of glycogen granules within the detrusor adjacent to the urothelium). Fifty patients and 20 controls were included. Increased glycogen deposition was observed in 37 (74%) and 2 (10%) patients in the Study and Control Group, respectively (p < 0.01, OR 25.6, 95% CI 5.2-125.8). In the subgroup analysis, no statistically significant difference was found between glycogen deposition score and IPSS, maximum detrusor pressure at maximum flow (PdetQmax) and duration of LUTS. In multivariate logistic regression, history of retention was the only variable which could predict high glycogen deposition (p = 0.019). Our results demonstrate increased detrusor glycogen deposition in patients with BOO due to BPH, but the amount of deposition did not seem to correlate with symptom severity and duration or urodynamic findings.

Mixed neuroendocrine-non-neuroendocrine neoplasm of the gallbladder: case report and literature review

BackgroundMixed neuroendocrine–non-neuroendocrine neoplasms (MiNENs) of the gallbladder are rare malignancies. Here we presented two cases and reviewed the related literature.Case presentationOur two patients were postoperatively diagnosed with gallbladder MiNENs, which pathologically consisted of a large cell neuroendocrine carcinoma and papillary adenocarcinoma. After cholecystectomy, one patient had a survival time of 30 months, while the other remained alive through 12 months of follow-up. In the literature, a total of 72 cases of gallbladder MiNENs were identified, and with our two patients included, we calculated a male-to-female ratio of 0.22 and a mean age of 64.5 years for the 74 reported cases. About one-half of these patients were found to have gallstones and presented with abdominal pain or discomfort in a relatively early stage. The preoperative diagnosis of these 74 cases mainly relied on abdominal ultrasound, contrast-enhanced computed tomography (CT) scanning, and magnetic resonance imaging or positron emission tomography/CT. However, the final diagnosis was established based upon the pathological evidence and expression of synaptophysin (Syn) and/or chromogranin A identified by immunohistochemical staining or neurosecretory granules detected by electron microscopy. Fifty-eight patients (78.4%) underwent various operations including simple cholecystectomy (n = 14), en bloc cholecystectomy (n = 9), standard or non-standard radical cholecystectomy (n = 25), or extended radical cholecystectomy (n = 6). The mean size of the resected gallbladder masses was 50.8 ± 36.1 mm (n = 63) with regional lymph node metastasis in 37 patients (52.1%), liver invasion or staging greater than T3 in 33 patients (45.8%), and hepatic metastasis in 26 patients (35.1%). The postoperative median survival time was 36 ± 11.42 months (95% confidence interval, 13.62 to 58.38 months). The log-rank analysis did not find that postoperative adjuvant chemotherapy contributed to a longer survival time relative to that among the patients who did not receive chemotherapy (numbers of patients, 15 versus 43; survival times, 36 months versus 30 months, p > 0.05).ConclusionsOur two cases and the cases in the literature suggest that MiNENs of the gallbladder predominantly occur in women; are associated with early lymph node metastasis, local hepatic invasion, and hepatic metastasis; and can be managed by various surgeries as well as chemotherapy combined with somatostatin analogs.

Modified Giemsa Stain: A solution to improve the quality of hypercellular bone marrow smears.

Morphology is still the cornerstone of the diagnosis of haematological malignancies where the quality of staining plays a major role. The hypercellularity of a smear interferes with the quality of staining. We compared a newly modified giemsa stain (MGS) as a routine method for staining hypercellular bone marrow smears over the conventional Leishman method and the May Grunwald Giemsa method (MGG) in a completely randomized block study. Quality of staining, cost, labour intensiveness and turnaround time of the new method was compared with MGG and Leishman methods for bone marrow smears of acute leukaemia using 30 cases. Bone marrow smears were blindly reviewed for the degree of staining of nuclei, cytoplasm and granules on a scale of 0 to 4 and statistically analysed by Minitab 16. A significant difference (p<.05) was revealed for the quality of staining of nuclei, cytoplasm and granules, and it was found that the new MGS was superior to the MGG and Leishman method. The cost of staining was lowest in the new MGS and the staining time of the new MGS was 76% less than MGG and 9.09% greater than the Leishman method. In terms of turnaround time, the new MGS is somewhat similar to the Leishman method and is user-friendly with a lesser number of steps to follow, compared with MGG. Our study showed that the new MGS is overall far superior, cost-effective and user-friendly compared with other conventional staining methods when used for hypercellular bone marrows.

In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. KEY POINTS: • A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress.

Solitary cutaneous hemorrhagic bullous mastocytoma: A rare entity

Solitary cutaneous mastocytoma, a clinical type of cutaneous mastocytosis, may present from birth itself as a macule, plaque, nodule, or bulla. It may be associated with pruritus, flushing attacks, or convulsions. The diagnosis of a solitary mastocytoma is made by the presence of a characteristic skin lesion and confirmed by typical histopathological features of mast cell clusters in dermis and subcutaneous tissues, metachromatic staining of mast cell granules with toluidine blue or giemsa staining, and immunohistochemical mast cell marker c-kit/ CD-117 staining. We report a 4-month-old child who presented with a hemorrhagic bulla of left knee. Skin biopsy and immunohistochemistry confirmed the clinical diagnosis of solitary cutaneous mastocytoma. We did not come across any previous report of solitary cutaneous mastocytoma presenting as hemorrhagic bulla.

Bushen-Huatan-Yizhi formula reduces spatial learning and memory challenges through inhibition of the GSK-3β/CREB pathway in AD-like model rats

BackgroundThere is an increase in cases of Alzheimer's disease (AD) stemming from a globally ageing population demographic. Although substantial research efforts were performed for the scope of prophylaxis and therapeutic measure development against AD, based on its pathogenesis, most were unsuccessful. Bushen-Huatan-Yizhi formula (BSHTYZ) is extensively implemented to manage dementia. However, few studies have been carried out to understand how BSHTYZ enhances recovery of spatial learning and memory and how it modulates relevant molecular interplays in order to achieve this. PurposeTo investigate neuroprotective function, ameliorating learning/memory capacity of BSHTYZ via GSK-3β / CREB signaling pathway in rat AD models influenced through Aβ1-42. MethodsA total of 60 male SD rats (3 months old) were randomized into six groups and treated with 2.6 μg/μl Aβ1-42 (5 μl) into the lateral ventricle, though the control group (Con) was administered an equivalent volume of vehicle. Consequently, the rat cohorts were administered either BSHTYZ or donepezil hydrochloride or normal saline, by intragastric administration, for four weeks. Spatial learning / memory were detected through the Morris water maze, and possible mechanisms detected by histomorphological examination and Western blot in the rat AD models induced by Aβ1-42. ResultsSpatial learning/memory issues were monitored after Aβ1-42 infusion in rats. Simultaneously, neuron loss in cornuammonis1 (CA1) / dentate gyrus (DG) within hippocampus region were identified, together with enhanced black granule staining within the hippocampus and hyperphosphorylated tau within Ser202 and Ser396 sites. It was also elucidated that Aβ1-42 had the capacity to up-regulate glycogen synthase kinase-3β (GSK-3β) and down-regulate cAMP response element binding protein (CREB). BSHTYZ was found to reverse such molecular interplays. ConclusionThe study suggested BSHTYZ could possibly provide neuroprotective role against learning / memory impairment, which provided a potential therapeutic tool delaying the progression of AD molecular interplays that includes the GSK-3β / CREB signaling pathway.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

Інформаційна технологія оцінки кольорових зображень метахромазії волютинових гранул дріжджів Saccharomyces cerevisiae УКМ Y-517

The research is devoted to the development of color imaging information technology, which is relevant for evaluating the results of cytochemical research in both biology and medicine. The aim of this work was to study the validity of software algorithms and integrated information technology for the qualitative and quantitative analysis of metachromasia reaction of volutin granules of Saccharomyces cerevisiae UCM Y-517 yeast. Methods. The object of this study was Saccharomyces cerevisiae UCM Y-517 yeast from the Ukrainian collection of microorganisms. The yeast was cultivated for 24 hours at 28oC. The metachromatic reaction of volutin granules was detected microscopically by the preparation of yeast cells stained with methylene blue. Digital images of cells were transferred to a specialized web resource for computer processing. Results. The algorithm of computer analysis of images included 3 steps: (1) recognition of single volutin granules, (2) assessment of the magnitude of metachromasia for each granule, and (3) integral estimation of the magnitude of metachromasia for the whole image. Information technology was developed and tested to support the daily monitoring of the metachromasia phenomenon, including obtaining the digital images, their transfer to a remote server, and automatic processing. Information technology is integrated into the web portal of the large-scale biophysical experiment “Heliomed”. Conclusions. The proposed method of computer image processing gave a quantitative and qualitative assessment in the automatic mode of metachromatic staining of volutin granules and confirmed the previously proposed classification of this phenomenon on the basis of visual examination.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients.

Simple SummaryThe expression of the androgen receptor (AR) and its splice variant AR-V7 is crucial for prostate cancer (PCa) biology. An immunohistochemical staining was performed on a tissue microarray with specimens from 410 PCa patients. AR staining, neither in the nucleus nor in the cytoplasm was associated with prognosis. AR-V7 staining of the general cytoplasm was associated with a shorter relapse free survival (RFS), whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. Further subgroup stratification for AR-V7 granular staining revealed it as an independent prognostic factor in younger patients (age ≤ 65), patients with negative CK20 staining and patients with perineural invasion. Altogether, AR-V7 protein detected in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

Isolation and Characterization of Lipid-Producing Bacillus sp. TC14

In order to obtain microbes producing microbial oil from environment, a strain, marked as TC14 was isolated by replica plating and Sudan Black staining on the selected media. Its colony is smooth; milk white, slightly transparent, round edge, sticky. Its cell is long-rod, G-, intracellular lipid granule. The results of physiological and biochemical research: catalase, denitrification and fluorescent pigment tests are negative; metachromatic granule staining, amylolysis, lecithinase tests are positive. According to its sequencing result of 16S rDNA, strain TC14 is classified and belonged to Bacillus, namely sp. TC14. The optimum inoculation temperature is 35°C and the most suitable initial pH value is 10. The intracellular lipid of strain TC14 is extracted by the way of hydrochloric acid hydrolysis and the calculated ratio of oil-production is 5.08%.

Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4'6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS).

Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4’6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 μg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.

Isolation and Characterization of Phosphorus Solubilizing Bacteria With Multiple Phosphorus Sources Utilizing Capability and Their Potential for Lead Immobilization in Soil

Phosphorus solubilizing bacteria (PSB) can promote the level of plant-absorbable phosphorus (P) in agro-ecosystems. However, little attention has been paid to PSB harboring abilities in utilizing multiple phosphorus sources and their potentials for heavy metal immobilization. In this study, we applied the strategy of stepwise acclimation by using Ca3(PO4)2, phytate, FePO4, and AlPO4 as sole P source. We gained 18 PSB possessing abilities of multiple P sources utilization, and these bacteria belonged to eight genera (Acinetobacter, Pseudomonas, Massilia, Bacillus, Arthrobacter, Stenotrophomonas, Ochrobactrum, and Cupriavidus), and clustered to two apparent parts: Gram-positive bacteria and Gram-negative bacteria. The isolate of Acinetobacter pittii gp-1 presented good performance for utilizing Ca3(PO4)2, FePO4, AlPO4, and phytate, with corresponding P solubilizing levels were 250.77, 46.10, 81.99, and 7.91 mg/L PO43–-P, respectively. The PSB A. pittii gp-1 exhibited good performance for solubilizing tricalcium phosphate in soil incubation experiments, with the highest values of water soluble P and available P were 0.80 and 1.64 mg/L, respectively. Additionally, the addition of A. pittii gp-1 could promote the immobilization of lead (Pb), and the highest Pb immobilization efficiency reached 23%. Simultaneously, we found the increases in abundances of both alkaline phosphatase gene (phoD) and β-propeller phytase gene (bpp) in strain gp-1 added soils. Besides, we observed the expression up-regulation of both pyrroloquinoline quinone gene (pqq) and polyphosphate kinases gene (ppk), with the highest relative expression levels of 18.18 and 5.23, respectively. We also found the polyphosphate particles using granule staining. To our knowledge, our findings first suggest that the solubilizing of tricalcium phosphate by phosphorus solubilizing bacterium belonging to Acinetobacter is coupled with the synthesis of polyphosphate. Taken together, A. pittii gp-1 could be a good candidate in improving soil fertility and quality.

Abstract 1421: Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells. METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo. CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC. Citation Format: Teruki Shimizu, Mako Tomogane, Yusuke Sano, Daiki Shimizu, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara. Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1421.

A Synthetic Pro-Drug Peptide Reverses Amyloid-β-Induced Toxicity in the Rat Model of Alzheimer's Disease.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder, involves the formation of the extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles. The current therapies against AD are symptomatic with limited benefits but associated with major side effects. Inhibition of self-aggregation of Aβ peptides into higher order cross-β structure is one of the potential therapeutic approach which may counter oligomerization of Aβ peptide. The present study aimed to evaluate the neuroprotective and anti-inflammatory potential of a synthetic Pro-Drug type peptide (PDp) against Aβ-induced toxicity in rat model of AD. Intra-hippocampal microinjection of toxic Aβ40 (IHAβ40) by stereotaxic surgery was performed in the male Sprague-Dawley rats to generate an Aβ-induced AD model. Sub-chronic toxicity of synthetic PDp using hematological, biochemical, and histopathological parameters was investigated. Evaluation of PDp on Aβ-induced neurodegeneration and neuroinflammation was performed. PDp inhibits plaque formation with increase in Nissl granule staining in the rat hippocampus. Aβ-induced toxicity associated imbalance in reactive oxygen species and antioxidant enzymes activity such as superoxide dismutase and catalase in the rat brain was overcome by PDp treatment. Tau protein hyperphosphorylation was normalized with PDp treatment. Also, the neuroinflammatory response was suppressed with PDp treatment. The present study depicts the potential neuroprotective role of PDp against Aβ-induced toxicity in rat. PDp inhibits plaque formation thereby normalizing oxidative stress, inhibiting tau protein hyperphosphorylation, and suppressing neuroinflammatory responses. Future studies done in this direction will pave way for new therapeutic strategies.

Tanshinone IIA Ameliorates Spatial Learning and Memory Deficits by Inhibiting the Activity of ERK and GSK-3β.

Alzheimer disease (AD) is the most common type of dementia which is becoming a primary problem in the present society, but it lacks effective treatment methods and means of AD. Tanshinone IIA (Tan IIA) has been reported to have neuroprotective effects to restrain the Aβ25-35-mediated apoptosis. However, few studies try to understand how Aβ1-42 affects hyperphosphorylation of tau and how Tan IIA regulates this process at the molecular level. Fifty male Sprague-Dawley rats were randomly divided into 5 groups and infused through the lateral ventricle with Aβ1-42 except the control group. Then the rats were treated with Tan IIA through intragastric administration for 4 weeks. After the ability of learning and memory being measured, histomorphological examination and Western blot were used to detect the possible mechanism in the AD-associated model rats. We observed that Aβ1-42 infusion could induce spatial learning and memory deficits in rats. Simultaneously, Aβ1-42 also could reduce the neuron in cornu ammonis 1 and dentate gyrus of hippocampus, as well as increase the levels of cleaved caspase 3, hyperphosphorylated tau at the sites Ser396, Ser404, and Thr205 with enhancing staining of black granules in brain. We also found that Aβ1-42 could increase the activity of extracellular signal-regulated protein kinase (ERK) and glycogen synthase kinase-3β (GSK-3β). Meanwhile, these phenomena could be ameliorated when Tan IIA was used. We concluded that Tan IIA might have neuroprotective effect and improving learning and memory ability to be a viable candidate in AD therapy with mechanisms involving the ERK and GSK-3β signal pathway.