Higher Cell Numbers Research Articles

Spontaneous Lethal Outbreak of Influenza A Virus Infection in Vaccinated Sows on Two Farms Suggesting the Occurrence of Vaccine-Associated Enhanced Respiratory Disease with Eosinophilic Lung Pathology.

Influenza A virus (IAV) infections in swine are usually subclinical, but they can reach high morbidity rates. The mortality rate is normally low. In this study, six vaccinated, spontaneously deceased sows revealed IAV infection and enhanced neutrophilic bronchopneumonia with unexpectedly large numbers of infiltrating eosinophils. The purpose of this study was to characterize these lung lesions with special emphasis on the phenotypes of inflammatory cells, the presence of eosinophilic peroxidase (EPO), and neutrophil extracellular traps (NETs). The number of Sirius red-stained eosinophils was significantly higher in the lungs of IAV-infected sows compared to healthy pigs, indicating a migration of eosinophils from blood vessels into the lung tissue stimulated by IAV infection. The detection of intra- and extracellular EPO in the lungs suggests its contribution to pulmonary damage. The presence of CD3+ T lymphocytes, CD20+ B lymphocytes, and Iba-1+ macrophages indicates the involvement of cell-mediated immune responses in disease progression. Furthermore, high numbers of myeloperoxidase-positive cells were detected. However, DNA-histone-1 complexes were reduced in IAV-infected sows, leading to the hypothesis that NETs are not formed in the IAV-infected sows. In conclusion, our findings in the lungs of IAV-infected vaccinated sows suggest the presence of so far unreported field cases of vaccine-associated enhanced respiratory disease.

Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p &amp;lt; 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

Selection versus transmission: Quantitative and organismic biology in antibiotic resistance

We aimed to determine the importance of selection (mostly dependent on the anthropogenic use of antimicrobials) and transmission (mostly dependent on hygiene and sanitation) as drivers of the spread of antibiotic-resistant bacterial populations. The first obstacle to estimating the relative weight of both independent variables is the lack of detailed quantitative data concerning the number of bacterial cells, potentially either pathogenic or harmless, and bacterial species exposed to antimicrobial action in the microbiotas of specific environments. The second obstacle is the difficulty of considering the relative importance of the transmission and selection exerting their combined effects on antibiotic resistance across eco-biological levels. As a consequence, advances are urgently required in quantitative biology and organismic biology of antimicrobial resistance. The absolute number of humans exposed to antibiotics and the absolute number of potentially pathogenic and commensal bacteria in their microbiomes should influence both the selection and transmission of resistant bacterial populations. The “whole Earth” microbiome, with astonishingly high numbers of bacterial cells and species, which are also exposed to anthropogenic antimicrobials in various biogeographical spaces, shapes the antibiotic resistance landscape. These biogeographical spaces influence various intensities of selection and transmission of potentially pathogenic bacteria. While waiting for more precise data, biostatistics analysis and mathematical or computational modeling can provide proxies to compare the influence of selection and transmission in resistant bacteria. In European countries with lower sanitation levels, antibiotic consumption plays a major role in increasing antibiotic resistance; however, this is not the case in countries with high sanitation levels. Although both independent variables are linked, their relative influence on the level of antibiotic resistance varies according to the particular location. Therefore, interventions directed to decrease antibiotic resistance should be designed “a la carte” for specific locations with particular ecological conditions, including sanitation facilities.

O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes

Abstract Introduction and aims Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state. Methods Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions. Results Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy. Conclusions Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.

Liver-specific Bmal1 deletion in mice leads to increased aortic stiffness and altered vascular remodeling

Circadian rhythms have an important role in cardiovascular physiology. Circadian clock genes are critical for vascular homeostasis. Global loss of the circadian clock gene Bmal1 increases aortic stiffness with impaired vascular function and blood pressure rhythm in mice. We previously found that hepatocyte-specific Bmal1 deletion (HBK) in the liver changes perivascular adipose tissue-mediated vascular function in young adult mice, indicating that liver circadian clock disruption distally affects function in another tissue and potential cross-talk between the liver and vasculature. We hypothesized that liver-specific Bmal1 deletion leads to aortic stiffness and pathological vascular remodeling. Studies were performed in 4- to 6- month old male HBK and flox control mice. Aortic stiffness, measured by pulse wave velocity, was significantly elevated in HBK mice compared to flox control mice (Flox: 1.84 ± 0.1 m/s; HBK: 2.9 ± 0.2 m/s; n = 5, p &lt; 0.01). Systolic blood pressure (tail-cuff) in the light phase was similar in both flox control and HBK mice (Flox: 101 ± 1 mm Hg; HBK: 103 ± 2 mm Hg; n = 3-5, p = 0.35). Aortas and plasma were collected at ZT2 for histological analysis and metabolite measurements. MetaMorph software analysis of Masson’s Trichrome-stained aortic sections revealed significantly increased aortic wall fibrosis in HBK mice (% fibrosis per area, Flox 17.0% ± 3.3%, HBK 27.4% ± 2.1%, p=0.03). The medial cell number in H&amp;E stained-aortic sections was increased in HBK mice compared to flox control mice (Flox: 534.6 ± 31.9; HBK: 1069 ± 90.1; p &lt; 0.01). TUNEL stained apoptotic cells were also counted and more apoptotic cells were observed in young HBK mice aortas compared to flox controls (Flox: 224.4 ± 56.1; HBK: 408.4 ± 20.8; p = 0.02). Aortic wall thickness was not different in young adult HBK mice compared to flox control mice. Aortic levels of the lipid peroxidation marker vascular 4-hydroxynonenal (4-HNE), a measure of oxidative stress, were similar between flox control and HBK mice. In plasma, the oxidative stress marker, 8-hydroxy-2’-deoxyguanosine (8-OHdG), a DNA damage product, and plasminogen activator inhibitor-1 (PAI-1) were not different between genotypes. In conclusion, our findings indicate that liver circadian clock disruption increases aortic stiffness and fibrosis with higher cell numbers as well as aortic apoptosis. These data suggest that altered vascular remodeling in HBK mice may contribute to aortic stiffness resulting in cardiovascular disease. Funded by P01HL136267 to JSP, R21AA026906 to SMB, and AHA 856877 to PP. 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.

Nanostarch-Stimulated Cell Adhesion in 3D Bioprinted Hydrogel Scaffolds for Cell Cultured Meat.

Three-dimensional (3D) bioprinting has great potential in the applications of tissue engineering, including cell culturing meat, because of its versatility and bioimitability. However, existing bio-inks used as edible scaffold materials lack high biocompatibility and mechanical strength to enable cell growth inside. Here, we added starch nanoparticles (SNPs) in a gelatin/sodium alginate (Gel/SA) hydrogel to enhance printing and supporting properties and created a microenvironment for adherent proliferation of piscine satellite cells (PSCs). We demonstrated the biocompatibility of SNPs for cells, with increasing 20.8% cell viability and 36.1% adhesion rate after 5 days of incubation. Transcriptomics analysis showed the mechanisms underlying the effects of SNPs on the adherent behavior of myoblasts. The 1% SNP group had a low gel point and viscosity for shaping with PSCs infusion and had a high cell number and myotube fusion index after cultivation. Furthermore, the formation of 3D muscle tissue with thicker myofibers was shown in the SNP-Gel/SA hydrogel by immunological staining.

TYPOLOGIES OF LAND USE AND LAND COVER ASSOCIATED WITH CASES OF CUTANEOUS LEISHMANIASIS IN A METROPOLITAN REGION IN THE BRAZILIAN AMAZON

Characterizing the probable site of infection of cutaneous leishmaniasis (CL) in face of anthropic changes in the Amazon makes it possible to understand the distribution of this disease in order to adopt control measures. The objective of this study is to identify landscape typologies resulting from changes in land use and land cover in the Metropolitan Region of Santarém regarding the occurrence of CL cases in 2012 and 2014. Landscape typologies were developed from TerraClass project data using regular 1 km² cells. Landscape presence and dominance metrics were used to generate cells with a single class and cells with more than one class, called mosaics. For cases of leishmaniasis, the metric was the presence of the disease. Association analyses were extracted from a 2x2 contingency table. The primary forest typology (PP04) had the highest number of cells in both years analyzed. However, changes in land use and land cover were evidenced by the growth in the number of cells with mosaics of agriculture (PP11 and PP12), urbanized areas (PP03 and PP10), and pastures (PP13). The presence of at least one case of CL in each year occurred in ten typologies, particularly in compositions with urbanized areas, pastures, and secondary vegetation. Typologies with the agriculture class, although the number of cells increased, did not follow the same growth logic regarding the presence of the disease. This study makes it possible to identify and characterize the places where CL occurs and provides further information for health surveillance agencies

Effect of Day 3 cell number on the live birth rate of vitrified-warmed Day 5 single blastocyst transfer in young women.

Previous studies have reported inconsistent results regarding blastocyst selection with a high day 3 (D3) cell number and the eventual pregnancy outcomes. Thus, in this study, the relationship between the D3 cell number and clinical outcomes of day 5 single blastocyst transfer (SBT) in vitrified-warmed transfer cycles was investigated. Our retrospective study included 1144day 5 SBT in vitrified-warmed cycles between February 2016 and February 2021. All cycles were the first vitrified-warmed cycles, and the female patients were less than 35 years of age. Based on the D3 cell number, the cycles were divided into four groups, as follows: group A (3-7 cells, n = 130); group B (8-9 cells, n = 621); group C (10-12 cells, n = 328); and group D (13-16 cells, n = 65). The differences in the live birth rate (LBR), clinical pregnancy rate, and miscarriage rate were examined among the four groups. The LBR and clinical pregnancy rate increased with the D3 cell number (P < 0.01). No significant difference was found in the miscarriage rate among the groups (P = 0.055). After adjusting for confounding factors, the LBR was significantly higher in groups C (odds ratio [OR] = 1.477, 95% confidence interval [CI]: 1.124-1.941, P = 0.005) and D (OR = 2.000, 95% CI: 1.166-3.429, P = 0.012) than in group B. A high D3 cell number (> 9 cells) was associated with a high LBR in the vitrified-warmed day 5 SBT cycles of patients < 35 years of age. The cell number of D3 embryos can be an important reference indicator for blastocyst selection. Among blastocysts with the same morphological score, those with > 9 cells on D3 can be preferentially selected for transplantation.

Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

The Expression of METTL14 in Patients with Newly Diagnosed Acute Myeloid Leukemia and Its Clinical Significance

To detect the expression of RNA methyltransferase 14(METTL14) in bone marrow of patients with newly diagnosed acute myeloid leukemia (AML), and to investigate the clinical and prognostic significance of METTL14 expression in newly diagnosed AML. Bone marrow samples were collected from 100 patients with newly diagnosed AML as observation group and 60 patients with iron deficiency anemia AML as control group. And collected the clinical data of the AML patients. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of METTL14 in AML and IDA patients. The relationship between the expression level of METTL14 and clinicopathological features, prognosis was analyzed. Kaplan-Meier curves were used to analyze the effect of METTL14 on overall survival (OS) in AML patients. Cox risk regression model was used to analyze the prognostic factors affecting in patients with AML. Compared with the control group, the expression of METTL14 was significantly increased in AML patients (P < 0.05). Compared with the METTL14 low-expression group, patients in the METTL14 high-expression group had advanced age, high bone marrow cell number, poor efficacyand poor prognosis(P < 0.05). The overall survival time of patients with the METTL14 high-expression group was significantly shorter than that of the low-expression group (P < 0.05). The high expression of METTL14 was an independent risk factor for poor prognosis in AML. METTL14 is significantly overexpressed in AML patients, and its correlated with poor clinicopathological features and poor prognosis. It can be used as a prognostic marker and potential therapeutie target for AML patients.

Abstract 332: Improved high-throughput genomic analysis of urine samples using pixelated-ultrasound to enable personalized genomic profiling and therapeutic developments in cancer

Abstract Here we address some of the existing challenges in liquid biopsy workflows which can potentially benefit urine analysis from bladder cancer patients because approximately 1/4th of all human cancers are urological tumors. Globally, bladder cancer is the 10th most prevalent, and the 9th cause of mortality due to malignancy. The challenge addressed here is an improvement to sample preparation for sequencing. Among genders and disease states, urine samples vary in terms of cell count and hence, in DNA concentration. Therefore, it is critical to obtain consistent DNA fragments from variable samples across large cohorts of patients so that the sequencing leads to accurate identification of biomarkers of disease risk, progression, and treatment-response. In this direction, we present the advantages of a novel pixelated ultrasound technology which significantly improves sample-preparation in a very fast, economical, and high-throughput manner. Here, DNA is extracted from human urine samples and subjected to sonication to generate fragment sizes. Extracted DNA was loaded onto a regular 96-well round-bottom plate, which is sealed and placed inside the system where 12 columns of the 96-well plate are individually programmed and run simultaneously, hence enabling quick optimization. The system does not require time- and labor-intensive processing of the samples before or after the sonication which is required for other similar technology. Overall, the DNA samples were processed with significant improvement over other similar technology in terms of consistency, speed, and economy. Subsequently, the fragment sizes were analyzed. Analysis of results, as presented in the figures, show that the DNA fragment had the average size as predicted, uniform shearing, and very consistent across the 96-well plate containing different samples with no cross-contamination. The time, labour, expense, and risk turned out to be very low compared to other similar technology. The sizes were appropriate for library preparation and sequencing. The technology works consistently for both low and high cell numbers which is a critical factor for heterogenous samples from patients. In conclusion, the study presents improvements to library preparation for sequencing which fit into the larger workflow of non-invasive liquid biopsy using urine samples. The ability to obtain consistent DNA fragments in a quick and economical manner will greatly enable unique fingerprinting of an individual's cancer or cancer risk across large cohorts, with the potential for integration in automation workflows. Citation Format: Martha Evans-Holm, Nicolai Lumbres, Rwik Sen, Patricia Montilla-Perez, Marc A. Paradise, Peter Lentz. Improved high-throughput genomic analysis of urine samples using pixelated-ultrasound to enable personalized genomic profiling and therapeutic developments in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 332.

Integrative analysis of homologous recombination repair patterns unveils prognostic signatures and immunotherapeutic insights in breast cancer

Globally, breast cancer (BC) is the leading cause of female death and morbidity. Homologous recombination repair (HRR) is critical in BC. However, the prognostic role and immunotherapy response of HRR in BC remains to be clarified. Firstly, we identified HRR types in BC samples from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) dataset (GSE42568) based on 65 HRR genes (HRRGs). A differentially expressed gene (DEG) list for different HRR types was generated. Then, the influences of gene sets composed of these DEGs on biological pathways and BC prognosis were explored. Next, we identified gene clusters based on gene sets composed of DEGs. Genes associated with prognosis for DEGs were identified using univariate Cox regression. Finally, the HRR score was constructed based on genes associated with prognosis. We analyzed how HRR score correlates with tumor mutation burden (TMB), immune cell infiltration (ICI), and immunotherapy response. Three HRR clusters were discovered. HRR subtype A demonstrated decreased infiltration and a high number of immunosuppressive cells with a poor prognosis. DEGs among various HRR types were predominantly enriched in cell cycle and genomic stability-related pathways. The prognostic model based on sixteen DEGs accurately predicted BC prognosis. The HRRGs were differentially expressed in three DEG clusters. TMB, ICI, and immunotherapy responses differed significantly between the high and low HRR groups (HSG, LSG). The HSG was distinguished by a high degree of ICI and low TMB. LSG had a better response to anti-PD-1 or anti-PD-1 and anti-CTLA4 combination therapy. This work revealed that HRR patterns would contribute to predicting prognosis and immunotherapy response in BC, which may benefit patients.

Hairy cell leukemia 2024: Update on diagnosis, risk-stratification, and treatment-Annual updates in hematological malignancies.

Hairy cell leukemia (HCL) and HCL-like disorders, including HCL variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL), are a very heterogenous group of mature lymphoid B-cell disorders characterized by the identification of hairy cells, a specific genetic profile, a different clinical course and the need for appropriate treatment. Diagnosis of HCL is based on morphological evidence of hairy cells, an HCL immunologic score of 3 or 4 based on the CD11c, CD103, CD123, and CD25 expression, the trephine biopsy which makes it possible to specify the degree of tumoral bone marrow infiltration and the presence of BRAFV600E somatic mutation. Progression of patients with HCL is based on a large splenomegaly, leukocytosis, a high number of hairy cells in the peripheral blood, and the immunoglobulin heavy chain variable region gene mutational status. VH4-34 positive HCL cases are associated with a poor prognosis, as well as HCL with TP53 mutations and HCL-V. Patients should be treated only if HCL is symptomatic. Chemotherapy with risk-adapted therapy purine analogs (PNAs) are indicated in first-line HCL patients. The use of chemo-immunotherapy combining cladribine (CDA) and rituximab (R) represents an increasingly used therapeutic approach. Management of relapsed/refractory disease is based on the use of BRAF inhibitors (BRAFi) plus R, MEK inhibitors (MEKi), recombinant immunoconjugates targeting CD22, Bruton tyrosine kinase inhibitors (BTKi), and Bcl-2 inhibitors (Bcl-2i). However, the optimal sequence of the different treatments remains to be determined.

RET Fusion Testing in Patients With NSCLC: The RETING Study

IntroductionRET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. MethodsThis situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. ResultsThe most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). ConclusionsIn-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Automated ChIPmentation procedure on limited biological material of the human blood fluke Schistosoma mansoni

In living cells, the genetic information stored in the DNA sequence is always associated with chromosomal and extra-chromosomal epigenetic information. Chromatin is formed by the DNA and associated proteins, in particular histones. Covalent histone modifications are important bearers of epigenetic information and as such have been increasingly studied since about the year 2000. One of the principal techniques to gather information about the association between DNA and modified histones is chromatin immunoprecipitation (ChIP), also combined with massive sequencing (ChIP-Seq). Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq in many model species, using in general roughly 106 cells per experiment. Such high cell numbers are sometimes difficult to produce. Using the human parasite Schistosoma mansoni, whose production requires sacrificing animals and should therefore be kept to a minimum, we show here that automated ChIPmentation is suitable for limited biological material. We define the operational limit as ≥20,000 Schistosoma cells with 30,000-300,000 cells as optimum. We also present a streamlined protocol for the preparation of ChIP input libraries.

Helicoid Morphology of Arthrospira platensis NIES-39 Confers Temperature Compensation in the Longitudinal Movement Velocity of Its Trichomes

The velocity of the gliding movement of filamentous cyanobacteria on a solid surface usually has a strong temperature dependency, and the higher the temperature, the faster the speed. Former studies on this phenomenon were conducted using filamentous cyanobacteria with straight morphology. We examined the velocity of the gliding movement of Arthrospira platensis NIES-39 along its longitudinal axis to see if the same was true for this cyanobacterium with helicoid trichomes. Experimental results showed little temperature dependency in the velocity in a wide temperature range in this cyanobacterium. However, when we examined the velocity using mutants with straight trichomes, their velocity was strongly affected by temperature, like other formerly analyzed filamentous cyanobacteria. This result indicates that the helicoid morphology of A. platensis trichomes confers temperature compensation to their migration velocity, enabling them to keep a relatively constant velocity under various temperatures. Migration of wild-type trichomes is considerably suppressed compared to the straight-trichome mutants on solid media. The temperature compensation in the locomotion of this organism appears to be established as part of such a suppression. It was also found that the velocity of this cyanobacterium depended on the trichome length when they were atypically short (&lt;250 µm); the shorter the trichomes, the slower the gliding movement tended to be. This result indicates that the coordinated action of a high number of cells constituting the trichome is required for efficient gliding movement.

Environmentally friendly assessment of ecological quality of watercourses based on banded demoiselle, Calopteryx splendens

AbstractDragonflies and damselflies are excellent bioindicators of the quality of both aquatic and terrestrial ecosystems. In our study, we assessed the usefulness of metric and meristic morphological traits as environmentally friendly and cost‐effective indicators of the integrative ecological quality of watercourses. Our study species of choice was the damselfly banded demoiselle, Calopteryx splendens (Harris) (Odonata: Calopterygidae). Adult C. splendens specimens were collected at three study sites along the trophic gradient of the Tisza River in Hungary and Ukraine. Body and abdomen length, head width, and distances between wing landmarks were assessed as metric traits, and the number of wing cells as meristic traits. The concentration of chlorophyll‐a was used to classify the three study sites into having a low, intermediate, and high trophic condition based on earlier studies. Significant differences were found along the trophic gradient based on the measured metric and meristic traits. Especially, insects from the site with the highest trophic condition had the largest body length, head width, and distances between wing landmarks, as well as the highest number of cells in the fore wings. There were also differences between males and females, but we did not find differences in fluctuating asymmetry (FA), i.e., differences between the left and the right wings. These results indicated that the morphological traits of adult banded demoiselles could be indicative of the environmental quality of a watercourse.

The response of human osteoblasts on bovine xenografts with and without hyaluronate used in bone augmentation

The aim of the in vitro study was to asses the effect of hyaluronate in conjunction with bovine derived xenografts on the viability, proliferation on day 4, 7 and 10, expression of early osteogenic differentiation marker Alkaline phosphatase on day 14 and 21, collagen, calcium deposition on day 14, 21 and 28 and cellular characteristics, as assessed through live cell image analysis, confocal laser scanning microscopy and scanning electron microscopy, in primary human osteoblasts compared to three bovine xenografts without hyaluronate. All experiments were performed in triplicates. Data were compared between groups and timepoints using one-way analysis of variance (ANOVA). Bonferroni post hoc test were further used for multiple comparison between groups (p < .05) An increase in cell viability (p < .05) and enhanced ALP activity was observed in all xenografts. Specimens containing hyaluronate showed a highest significant difference (23755 ± 29953, p < .0001). The highest levels of calcium (1.60 ± 0.30) and collagen (1.92 ± 0.09, p < .0001) deposition were also observed with hyaluronate loaded groups. The osteoblasts were well attached and spread on all xenograft groups. However, a higher number of cells were observed with hyaluronate functionalized xenograft (76.27 ± 15.11, (p < .0001) in live cell image analysis and they migrated towards the graft boundaries. The biofunctionalization of xenografts with hyaluronate improves their in vitro performance on human osteoblasts. This suggests that hyaluronate might be able to improve the bone regeneration when using such xenografts.

Distribution and Clinicopathological Features of Mott Cells (Plasma Cells Containing Russell Bodies) in Gastric Cancer: Presence of Mott Cells Is Associated with Favorable Prognosis

Gastric cancer (GC) is still one of the leading causes of cancer-related mortality. We previously reported the relationship between histological heterogeneity of tumor cells and molecular features in GC. The tumor microenvironment also has a crucial role in GC progression and therapeutic resistance. In this study, we focused on the tumor microenvironment, especially inflammatory cells in GC. Using GC tissue slides, we investigated the distribution and clinicopathological significance of inflammatory cell counts including eosinophils, neutrophils, lymphocytes, and plasma cells. Additionally, we investigated the relationship between Mott cells (plasma cells containing Russell bodies) and clinicopathological features. In neoplastic gastric mucosa, a high number of plasma cells was associated with low T-grade, early stage, and good prognosis. We then focused on Mott cells and found that their presence in neoplastic gastric mucosa was associated with lower T and N grades, early stage, and Helicobacter pylori infection and was inversely associated with CD44 and EGFR expression. Additionally, the presence of Mott cells was associated with good prognosis in advanced GC and was an independent favorable prognostic predictor. The presence of Mott cells in GC might be one useful prognostic predictor, and Mott cells might have an important role in the carcinogenesis of H. pylori infection.