Initial Characterization Research Articles

Initial characterization of immune microenvironment in pheochromocytoma and paraganglioma.

Due to fewer adverse events, faster onset of action, and longer durable responses compared to chemotherapy, immunotherapy has been widely used to treat advanced solid tumors. Moreover, immunotherapy can improve the autoimmune status, thus allowing patients to benefit from the treatment in the long term. The immune microenvironment status is closely associated with the response to chemotherapies. Here, we analyzed the characteristics of the immune microenvironment in pheochromocytoma and paraganglioma (PPGL). Immunohistochemistry showed that PD-L1 is sparely expressed in PPGL with low positive rates and low expression levels, an expression pattern, that is, not correlated with tumor malignancy. Moreover, the level of intratumoral CD4+ and CD8+ lymphocyte infiltration in PPGL is low, suggesting that the immune microenvironment in PPGL may be in "immune desertification" or "immune rejection" states in which CD4+ and CD8+ lymphocyte infiltration is prevented, rendering immunotherapy less effective. In sum, our results indicate that PPGL is a microsatellite-stable tumor with low tumor mutational burden (TMB) levels, weak neoantigen production, and poor tumor antigenicity, hinting at a poor response of PPGL to chemotherapies.

Characterization of visual response properties and connectivity of Wide-field vertical neurons in the mouse superior colliculus

Vision is a sense that serves different ethological functions across species. Visual information can be used to either detect preys or predators and generate orienting or escaping responses. The superior colliculus has been implicated in the orchestration of these behaviors. Within this structure, at the interface between the visual and the multisensory layers, lay the wide-field vertical neurons (WFV). WFV are a conserved morphologically defined cell-type found across many taxa from reptiles to highly visual mammals, independently of their visual ecology. In mammals, these neurons receive direct inputs from the retina and the primary visual cortex, and have the pulvinar as a known target, however, their brain-wide connectivity remains to be identified. These cells display peculiar response properties, but despite their ubiquity and unique morphology, their contribution to vision remains elusive. In this study, we have characterized WFV morphology, connection, and visual response properties in vivo in the laboratory mouse. Viral intersectional approaches revealed their projection patterns. Two-photon calcium imaging in awake mice using a transgenic Cre-line targeting specifically WFV (NTSR1-Cre) confirmed their unique response properties. WFV showed a strong preference for small moving stimuli and a slight bias for certain directions. Interestingly, visual responses to distinct visual stimuli showed variable degrees of adaptation upon repeated presentation. In addition, Cre-dependent rabies tracing unraveled a variety of sources of input which had not been described previously. These results place WFV as a major integrator of visual information in the superior colliculus. Their adaptation to looming stimulus suggest a role in ethologically relevant function in avoiding inappropriate responses, while sustained responses to small moving stimulus might be linked to attentional processes and orienting behaviors. Taken together, this initial characterization paves the way to elucidate the role of these neurons in visual behavior.

Evaluation of docking procedures reliability in affitins-partners interactions.

Affitins constitute a class of small proteins belonging to Sul7d family, which, in microorganisms such as Sulfolobus acidocaldarius, bind DNA preventing its denaturation. Thanks to their stability and small size (60-66 residues in length) they have been considered as ideal candidates for engineering and have been used for more than 10years now, for different applications. The individuation of a mutant able to recognize a specific target does not imply the knowledge of the binding geometry between the two proteins. However, its identification is of undoubted importance but not always experimentally accessible. For this reason, computational approaches such as protein-protein docking can be helpful for an initial structural characterization of the complex. This method, which produces tens of putative binding geometries ordered according to a binding score, needs to be followed by a further reranking procedure for finding the most plausible one. In the present paper, we use the server ClusPro for generating docking models of affitins with different protein partners whose experimental structures are available in the Protein Data Bank. Then, we apply two protocols for reranking the docking models. The first one investigates their stability by means of Molecular Dynamics simulations; the second one, instead, compares the docking models with the interacting residues predicted by the Matrix of Local Coupling Energies method. Results show that the more efficient way to deal with the reranking problem is to consider the information given by the two protocols together, i.e. employing a consensus approach.

Use of biosensors for rapid and sensitive detection of pesticides in food samples for food safety chemical risk assessment.

The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human health. For that reason, detection of pesticides and their biotransformation products in food is of utmost importance. According to previous studies, esterase-based biosensors have been proposed as a viable and efficient solution for the detection of organophosphate pesticides. In this project, a double mutant of the thermostable esterase-2 (EST2) from Alicyclobacillus acidocaldarius was studied as a potential biosensor, for its ability to detect residual amounts of pesticides. Initial characterisation of the enzyme was performed, that included determination of optimal pH, thermophilicity, as well as kinetic analysis. Subsequently, the enzyme was studied by enzymatic activity assays with and without the presence of various organophosphate compounds. The effect of the organophosphates on the enzymatic activity was measured and complete inhibition of the enzyme was observed after incubation with paraoxon. These experiments were followed by an additional method involving labelling of the enzyme with a fluorescent probe. In this case, the effect of different pesticides on the EST2 enzyme was monitored by measuring the fluorescence quenching upon addition to the enzyme. Fourteen compounds were screened with this method and significant fluorescence quenching was observed in the presence of paraoxon and methyl-paraoxon when used in equimolar amounts with the enzyme in the range of nanomolar. This biosensor has been also used to test the presence of pesticides in real food samples, like fruits and juices. This research represents a starting point to develop effective fluorescence-based biosensors aiming at the screening of mutants with different pesticide selectivity profiles. The use of this enzyme-based biosensor can have applications in the field of food traceability as well as environmental monitoring, to control the presence of toxic chemicals, in particular organophosphate pesticides.

APOE3 Christchurch mutation carriers from the Colombian kindred with autosomal dominant Alzheimer’s disease due to PSEN1 E280A

AbstractBackgroundOur understanding of the mechanisms of protection mediated by APOE has advanced significantly in the past years. Our group previously showed that APOE3 Christchurch (APOE3ch) homozygosity was associated with the protection against Alzheimer’s disease dementia in an individual who carried the Presenilin‐1 (PSEN1)‐E280A variant for autosomal dominant AD (ADAD) (Arboleda‐Velasquez et al., 2019). Here we present the initial characterization of individuals from the PSEN1‐E280A kindred with the APOE3ch variant.Methods1,000 PSEN1‐E280A variant carriers and 1,000 non‐carriers from the Colombian ADAD kindred were characterized in terms of their common APOE variants and the rare APOEch variant. Participants had previously completed a comprehensive neurological and neuropsychological evaluation. Those who were alive were re‐contacted in 2021 to undergo comprehensive clinical assessments, including the CERAD‐Colombia neuropsychological battery, and MRI and FDG PET imaging as part of the Resist Alzheimer’s Project.ResultsWe discovered 97 ADAD kindred members with the APOE3ch variant (95 heterozygotes), including 84 who are living and 13 who are deceased (7 who were brain donors). 29 of the APOE3ch carriers also had the PSEN1‐E280A variant, including 18 who are living and 11 who are deceased. The age range for all APOE3ch variant carriers was from 17 to 72 years, including 11 APOE3ch + PSEN1‐E280A carriers who were 50‐72 years old (i.e., older than the PSEN1‐E280A variant carriers’ median age at dementia onset). Overall, global cognition and memory performances of the first three APOE3ch + PSEN1‐E280A carriers (older than 50 years of age) assessed fell within the normal range, compared to age‐matched individuals from this kindred.ConclusionsThe prevalence rate of APOE3 Christchurch heterozygosity in the Colombian kindred with ADAD (4.85%) is very high compared to the general population (e.g., gnomAD database reports an allele frequency of 0.00001283; Karczewski et al., 2019). Preliminary clinical and cognitive data from APOE3ch variant carriers are consistent with the hypothesis that heterozygosity may have a mild‐to‐moderate protective effect against cognitive decline. We plan to compare longitudinal clinical, cognitive, biomarker, and post‐mortem neuropathological changes in PSEN1‐E280A carriers with and without the APOE3ch variant to clarify the role of APOE3ch heterozygosity in the protection from ADAD.

Initial Characterization of Bifidus Milk Quality with Different Starter Levels of Bifidobacterium longum

A study on the initial characterization of bifidus milk quality with a different level of Bifidobacterium longum was conducted to find out the best starter level that is able to produce bifidus milk that meets the characteristics of a healthy drink. The design use in this study consisting of 4 treatments (starter level) that are 1% (L1), 3% (L2), 5% (L3), and 7% (L4) with four replications for each treatment, thus 16 experimental units were obtained. Furthermore, the parameters measured were the degree of acidity (pH), total lactic acid, protein content, total plate count, and syneresis. The resulting data were statistically analyzed using ANOVA and Duncan’s multiple range test (DMRT) was performed if there was a difference. Results showed that the starting concentration had no effect on pH and total lactate, whereas had a highly significant effect on TPC and protein content (P < 0.01). However, all values are still within the range of Indonesian National Standards. Therefore, based on the results obtained, it is recommended that the highest quality bifidobacteria milk is indicated by pH, lactate content, protein, and total plate count obtained by L1 (1% starter) 3.84, 1.22, 4.92, and 8.40 respectively

Microscopy-Based Multiwell Assay to Characterize Disturbed Bacterial Morphogenesis Upon Antibiotic Action.

The urgent need of new antimicrobial agents to combat life-threatening bacterial infections demands the identification and characterization of novel compounds that interfere with new and unprecedented target pathways or structures in multiresistant bacteria. Here, bacterial cell division has emerged as a new and promising target pathway for antibiotic intervention. Compounds, which inhibit division, commonly induce a characteristic filamentation phenotype of rod-shaped bacteria, such as Bacillus subtilis. Hence, this filamentation phenotype can be used to identify and characterize novel compounds that primarily target bacterial cell division. Since novel compounds of both synthetic and natural product origin are often available in small amounts only, thereby limiting the number of assays during mode of action studies, we here describe a semiautomated, microscopy-based approach that requires only small volumes of compounds to allow for the real-time observation of their effects on living bacteria, such as filamentation or cell lysis, in high-throughput 96-well-based formats. We provide a detailed workflow for the initial characterization of multiple compounds at once and further tools for the initial, microscopy-based characterization of their antibacterial mode of action.

Resident macrophages of the lung and liver: The guardians of our tissues.

Resident macrophages play a unique role in the maintenance of tissue function. As phagocytes, they are an essential first line defenders against pathogens and much of the initial characterization of these cells was focused on their interaction with viral and bacterial pathogens. However, these cells are increasingly recognized as contributing to more than just host defense. Through cytokine production, receptor engagement and gap junction communication resident macrophages tune tissue inflammatory tone, influence adaptive immune cell phenotype and regulate tissue structure and function. This review highlights resident macrophages in the liver and lung as they hold unique roles in the maintenance of the interface between the circulatory system and the external environment. As such, we detail the developmental origin of these cells, their contribution to host defense and the array of tools these cells use to regulate tissue homeostasis.

Module-Level Thermal Interface Material Degradation in HALT

Abstract In this study, thermal interface material (TIM) degradation is driven through highly accelerated life test (HALT) using temperature cycling with a prescribed vibrational acceleration for two commercially available materials having thermal conductivities of 6.0 and 8.5 W/m K. HALT specimens were prepared by applying TIM through a 4-mil stencil over AlSiC baseplates in the shape of those used in Wolfspeed CAS325M12HM2 power electronics modules. Baseplates were mounted onto aluminum carrier blocks with embedded thermocouples to characterize the thermal resistance across the baseplate and TIM layer. Thermal dissipation into the top of the baseplates was provided by a custom heating block, which mimics the size and placement of the die junctions in CAS325 modules, applying power loads of 200, 300, and 400 W. After initial characterization, samples were transferred to the HALT chamber with one set of samples exposed to temperature cycling only (TCO) and the other temperature cycling and vibration (TCV). Both sample sets were cycled between temperature extremes of −40 °C and 180 °C with vibrations applied at a peak acceleration of 3.21 Grms. After hundreds of cycles, samples were reevaluated to assess changes in thermal resistance to provide an accelerated measure of TIM degradation. This allows for reliability prediction of useful lifetime (illustrated in a solar inverter case study herein), as well as to provide a basis for developing an accelerated testing method to related temperature cycling to faster methods of degradation. Such techniques provide a means to develop maintenance schedules for power modules for ensuring sufficient thermal performance over the operating lifetime.

Gelation-based feed-stock technologies for HVOF thermal spray development: Micro-composite powder preparation and HVOF coating microstructure

The current study proposes use of the polysaccharide, sodium alginate, as a gelation aid to ‘solidify’ aqueous suspensions, comprised of micron-sized ceramic-metal particles, into spherical, micro-composite ‘cermet’ feedstock powders. These feedstock powders, with finely divided ceramic and metallic components, can then be used for the preparation of high velocity oxygen fuel coatings and/or laser deposition cladding. Sodium alginate gelation is typically based on ion exchange, and a variety of gelation agents have been examined in the present work. It is shown that aqueous solutions of CaCl2, Ca-acetate, Al-nitrate, methanol, acetic acid, and 5 % HCl acid are all capable of gelling alginate droplets through a simple cross-linking mechanism. Consequently, spraying a sodium alginate containing ceramic-metal particle suspension, with a suitable air atomising nozzle, into an aqueous bath of one of the outlined solutions allows the production of nominally spherical ‘cermet’ feedstock particles. In the present work, TiC-Ni3Al micro-composite cermet particles have been successfully produced, and then analysed both before and after a sintering heat-treatment. Furthermore, rheological assessment of the colloidal suspensions both prior to and during the gelling stage are also described. As a preliminary ‘proof-of-principle’, an initial set of HVOF coatings has also been prepared using the gelled feedstock material, and initial characterisation of these coatings is presented.

How do we characterize temperament? Broad testing of temperament across time and contexts in low-variable conditions

In many species, individual animals often maintain relatively consistent individual differences in behaviour, indicative of temperament or personality. These stable individual differences are important for life history, survival and health consequences, and the field has seen exponential growth in temperament (e.g. boldness, exploration, sociability, aggression and activity) research. Quantification of animal temperament can be difficult because it requires (1) repeated behavioural measures from the same individual (to gauge within-individual consistency across time and contexts) and (2) identification of the most appropriate behaviour(s) to characterize temperament. In the current study, we took an atheoretical approach and conducted repeat, diverse behavioural tests in a low-variable environment to determine behaviours that were individually consistent over time and across conditions. To control environmental effects on behaviour, we conducted this work with individually housed outbred male Sprague–Dawley rats in a laboratory. To determine individual consistency, we measured each rat's behavioural response to five different arenas (i.e. different contexts) and repeated the five tests at three time points. To identify potential composite, multibehaviour estimates of temperament, we used factor analyses to determine behaviours that covaried on each test arena, and calculated factor scores. To determine individual consistency of behaviours and factor scores across time and contexts, we used reliability and correlation analyses. Several behaviours consistently covaried on each test, and with a few notable exceptions, the resulting factors tended to align with five commonly studied temperament categories. Certain behaviours/factors were more consistent across time and contexts than others; distance travelled/activity were highly consistent, as were sociality- and boldness-related behaviours, whereas aggression and exploration were less consistent. We did not find strong evidence that multibehaviour estimates of temperament (factor scores) were more consistent over time or across conditions than single-behaviour metrics. Where possible, similar atheoretical approaches to cross-time and cross-context behaviour testing will be beneficial for initial temperament characterization.

Redescription and molecular characterisation of Panagrolaimus labiatus (Kreis, 1929) Andrássy, 1960 (Rhabditida, Panagrolaimidae) from India and proposal of P. burdwanensis Chaturvedi &amp; Khera, 1979 as a junior synonym of P. labiatus

Summary Two populations of bacterial-feeding nematodes belonging to the genus Panagrolaimus were recovered from infected slugs collected from gardens, agricultural areas and nurseries of the Baghpat district (India). Initial morphological characterisation of these populations show that they resemble Panagrolaimus labiatus from China and Bulgaria, P. burdwanensis from India, and P. cf. labiatus from Iran. To clarify taxonomical affiliations, we morphologically compared these two new populations to those recovered from China, Bulgaria, India and Iran. In addition, we sequenced several taxonomically relevant genes and used them to reconstruct phylogenetic relationships. Our results show that the two Indian populations do not significantly differ morphologically from the specimens used to describe the species P. labiatus, and also do not differ among them. We only observed small variations in the size of males and females. In addition, morphologically, the nematodes used to describe P. burdwanensis are very similar to the nematodes used to describe P. labiatus and to the two populations described in this study. Therefore, we propose P. burdwanensis as a junior synonym of P. labiatus and that the two populations recovered from India belong to the P. labiatus species. Phylogenetic analyses using the nucleotide sequences of the internal transcribed spacer (ITS), 18S and 28S rRNA gene sequences support our morphological conclusions. Our study therefore provides a detailed morphological description and molecular marker 18S and 28S gene sequences that can support future taxonomical studies of this species and highlights the importance of using both molecular and morphological data for the proper description of soil microfauna.

Identification of a Novel Anti-HIV-1 Protein from Momordica balsamina Leaf Extract.

Our lab investigates the anti-HIV-1 activity in Momordica balsamina (M. balsamina) leaf extract. Traditional Senegalese healers have used M. balsamina leaf extract as a part of a plant-based treatment for HIV/AIDS infections. Our overall goal is to define and validate the scientific basis for using M. balsamina leaf extract as a part of the traditional Senegalese treatment. As an initial characterization of this extract, we used activity-guided fractionation to determine the active ingredient's solubility and relative size. We found that M. balsamina leaf extract inhibits HIV-1 infection by >50% at concentrations of 0.02 mg/mL and above and is not toxic over its inhibitory range (0-0.5 mg/mL). We observed significantly more antiviral activity in direct water and acetonitrile extractions (p ≤ 0.05). We also observed significantly more antiviral activity in the aqueous phases of ethyl acetate, chloroform, and diethyl ether extractions (p ≤ 0.05). Though most of the antiviral activity partitioned into the aqueous layers, some antiviral activity was present in the organic layers. We show that the active agent in the plant extracts is at least 30 kD in size. Significantly more antiviral activity was retained in 3, 10, and 30 kD molecular weight cutoff filters (p ≤ 0.05). In contrast, most of the antiviral activity passed through the 100 kD filter (p ≤ 0.05). Because the active anti-HIV-1 agent presented as a large, amphiphilic molecule we ran the purified extract on an SDS-page gel. We show that the anti-HIV-1 activity in the leaf extracts is attributed to a 30 kDa protein we call MoMo30. This article describes how MoMo30 was determined to be responsible for its anti-HIV-1 activity.

Innate Immune Microenvironment of Indolent B Cell Splenic Lymphomas: Role in Diagnosis and Treatment

Indolent splenic B-cell lymphomas are rare diseases (incidence <1:100,000) that include splenic marginal zone lymphoma (SMZL), hairy cell leukemia (cHCL), hairy cell leukaemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). These splenic lymphomas can have overlapping clinical presentations with only subtle differences in their morphology and phenotypes. However, there are considerable differences in prognosis, response to treatment, and outcomes. Better understanding of the tumor microenvironment (TME) is essential to design expedient and accurate diagnostic methods and precision drug treatments for each type of splenic B-cell lymphoma. We hypothesized each splenic lymphoma would have a distinct TME with some overlap for the red pulp lymphomas (cHCL, HCLv, and SDRPL). Human specimen collection and usage was approved by the Institutional Research Subjects Review Board. We studied cryo-preserved bulk splenic tumor suspension cell isolates from fresh (<4 hour) splenectomy tissue and intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells analyzed by high-parameter fluorescence flow cytometry with a 29-color myeloid panel using a Cytek Aurora spectral flow cytometer. Initial IHC characterization of the splenic TME illustrated a markedly different distribution of intratumoral T cells (CD3) and macrophages (Mθ) (CD163). In cHCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. Tissue architecture, as evidenced by splenic macrophage distribution, was markedly different. cHCL and HCLv showed diffuse but distorted networks of interdigitating macrophages, while SDRPL retained a nearly normal sinusoidal distribution of abundant macrophages, and SMZL showed total exclusion of CD163-positive Mθ from areas of confluent neoplastic white pulp. Analysis of splenic suspension cells showed an average of 72% B cells in the lymphoma specimens, twice that of trauma spleens. T cells made up the greatest fraction of non-B cells (~63%) for both the diseased and control samples. We analyzed the non-T and non-B cells to examine the innate immune cells. Granulocytes comprised on average 4% with no difference between the splenic lymphomas or trauma (Fig.1A). Trauma spleen had 86% macrophage/monocytes (M/Mθ)s, with similar values for HCLv (80%) and cHCL (74%). The percentage of M/Mθ was lower in SDRPL (55%) and SMZL (48%). We then analyzed the percentage of M/Mθ subtypes. As shown in (Fig.1B), CD14+CD16- was the major subtype for trauma (58%) and HCLv (50%). Conversely for cHCL, CD14loCD16+ was predominant (60%) subtype. SDRPL and SMZL shared similar subtype profiles with equal proportions of CD14+CD16- and CD14loCD16+ (40%). We then measured the dendritic cells of the innate immune population. SDRPL (41%) and SMZL (48%) had higher percentages than trauma (12%), HCLv (16%), or cHCL (20%). Conventional dendritic cell 2 (cDC2) was the predominant subtype comprising 50-74% of the DCs. Average conventional dendritic cell 1 (cDC1) and plasmacytoid dendritic cells (pDCs) were 30% and 9%, respectively. pDC was lower in cHCL than the other lymphomas (1%). Our data show a similar M/Mθ component of the innate immune TME in the red pulp lymphoma SDRPL and white pulp lymphoma SMZL. SDRPL and SMZL also had higher proportions of dendritic cells compared to cHCL, HCLv, or trauma spleens. cDC2 was the predominant DC type in the spleen regardless of disease state. We are currently analyzing flow cytometry panels to access natural killer cell distribution and T cell functionality. We aim to better understand immune cell interactions in the TME and discover variations between the splenic B-cell lymphomas, resulting in improved diagnosis and therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Initial Characterization of Nexi-001, a Donor-Derived T-Cell Product, for the Treatment AML Patients That Have Relapsed after Allogeneic Hematopoietic Stem Cell Transplant

Initial Characterization of Nexi-001, a Donor-Derived T-Cell Product, for the Treatment AML Patients That Have Relapsed after Allogeneic Hematopoietic Stem Cell Transplant

MODL-28. DEVELOPING A STRATEGY FOR MODELING HIGH-GRADE GLIOMA IN GӦTTINGEN MINIPIGS

Abstract BACKGROUND The current literature does not describe a reproducible large animal model of intracranial high-grade glioma (HGG). Prior work has demonstrated the feasibility of inducing HGG de-novo in rodents by targeting specific oncogenic pathways. Here we report our approach to the production of supratentorial HGG in a series of minipigs through lentiviral gene transfer and subsequent initial characterization of a porcine glioma cell line. METHODS Four minipigs received injections into the subcortical white matter using a combination of lentiviral vectors expressing platelet-derived growth factor beta (PDGF-B), HRAS, and shRNA-p53. Animals underwent behavioral monitoring through porcine neurobehavioral scoring (PNS) and veterinary monitoring. Magnetic resonance imaging (MRI) was conducted at endpoint prior to necropsy. Post-mortem tissue biopsies underwent tissue culture and neuropathologic evaluation with hematoxylin and eosin (H&amp;E) staining, immunohistochemistry, and immunofluorescent staining. Data are presented using appropriate statistical tests where relevant and descriptive statistics. RESULTS Two pigs received 50ul injections and reached endpoint by the end of post-operative week 1 and 2. Two pigs received 25 ul injections and were asymptomatic until a pre-determined endpoint of 4 weeks. MRI scans at endpoint demonstrated contrast enhancing, mass forming lesions at the site of injection with evidence of hemorrhage and perilesional edema, consistent with high-grade glioma. On H&amp;E staining high-grade glioma growth was identified in 100% of animals. We observed immunopositivity for tumor markers GFAP, OLIG2, NG2, SOX2, and PDGFRA, as well as redox markers, and microenvironmental features consistent with high-grade glioma. Porcine glioma cell cultures were found to have significantly greater proliferative rate compared to control, and demonstrated GFAP, OLIG2, PDGFRA, and CD68 immunopositivity. CONCLUSIONS Lentiviral gene transfer represents a feasible strategy for glioma modeling in the Gӧttingen minipig. With our described methodology, we present a realistic strategy for reproducible modeling of intracranial glioma as a platform for preclinical neurosurgical development programs.

Physical properties and processing of Silphium integrifolium seeds to obtain oil and enriched protein meal

AbstractSilphium integrifolium Michx. (Silflower) has been a promising subject for domestication as a perennial oilseed crop. This work was carried out to investigate the seed processing aspect of this effort. Thousand seed weight, densities, and dimension of the seeds were evaluated, seed milling to obtain enriched kernel fraction was conducted, and initial characterization of the seed protein was performed. There was wide variation in flat seed length (11.5–20.8 mm), width (4.6–11.8 mm), and thickness (0.9–1.6 mm). The thousand seed weight was 23.8 g but the tapped bulk density was only 189.6 g/L due to the presence of wing around the seed's periphery. The kernel accounted for 56.1% of the seed weight and contained 31.0% oil. A kernel‐enriched fraction with 79.6% purity was obtained by roller‐milling, sifting, and air classification. Linoleic (62.3%) and oleic (19.6%) acids were the major fatty acids in the oil. The defatted enriched kernel fraction contained 63.41% crude protein. Globulin, glutelin, albumin, and prolamin accounted for 55.6%, 19.3%, 16.4%, and 8.7% of the soluble proteins, respectively. At an extraction pH of 9, protein solubility was 62%. Maximum solubility (70%) was obtained at pH 10 while minimum solubility of 9% occurred between pH 4 and 5.5. Aside from the oil, the dehulling of silflower seeds also produced a high‐protein defatted meal, which may be used as is or as a starting material for enriching the protein further into a protein isolate.

Characterisation of Staphylococci Isolated from Milk Samples of a Water Buffalo Herd.

Water buffalo produce a tenth of milk for global human consumption. Non-aureus staphylococci (NAS) are among the most commonly isolated bacteria from mastitis in water buffalo and dairy cows. These results described the initial characterisation of 17 NAS-15 Staphylococcus simulans and two Staphylococcus chromogenes from a water buffalo herd (n = 44) in South Africa. The isolates were identified by classical microbiology, MALDI-TOF, and 16S rRNA, and the disc diffusion method determined the antibiotic susceptibility. A multi-locus sequence typing scheme (MLST) was developed to determine S. simulans sequence types (ST), by defining and comparing seven housekeeping gene fragment sequences. Sequence typing confirmed all 15 S. simulans isolates from water buffalo which belonged to a single ST, genetically distant from the six bovine STs isolated from adjacent farms, which also varied, indicating no current bacterial transfer between species. The antibiotic resistance patterns of S. simulans varied between beta-lactams. The mean milk somatic cell count (SCC) for the water buffalo milk samples was 166,500 cells/mL milk. This information offers insights into the epidemiology and comparison among isolates from various origins, which leads to effective proactive mastitis strategies resulting in safe, high-quality dairy products from water buffalo and dairy cows for human consumption.

Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants.

Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended.

Initial Results From the SPIDER Beamlet Current Diagnostic

Source for the production of ions of deuterium extracted from a radio frequency plasma (SPIDER) is the full-scale prototype for the ITER heating neutral beam (HNB) source. Beam operation in SPIDER, with the eventual aim of achieving 100-keV, 46-A hydrogen negative ion beam for 1 h, has mainly been performed in volume operation without cesium. Recently, with the introduction of cesium, SPIDER has moved to the surface operational regime, with a noted increase in beam current. Electrical and calorimetric measurements of the beam current have previously shown discrepancies between the two, possibly due to background gas interactions in the accelerator and upstream of the STRIKE calorimeter. Taking advantage of the reduced number of open apertures due to the plasma grid (PG) mask, a series of fluxgate current transducers have been installed in SPIDER to measure the current of individual beamlets. These beamlet current monitors (BCMs), positioned downstream of the grounded grid (GG) around individual grid apertures, have provided direct measurement of the current of up to five individual beamlets during the recent SPIDER campaigns with and without cesium. This contribution describes the installation of the BCM dc sensors, analysis of the dc signal, and initial characterization of the beamlet direct current component in volume and surface operational regimes.