A novel room concept for shared resource laboratories.

Shared resource laboratories/core facilities (SRLs) are centralized platforms that house and provide access to complex and expensive research equipment. Due to the highly complex nature of the instrumentation they support, SRLs have special environmental requirements for their laboratory space. Here, we describe the planning and establishment of a large light microscopy SRL, with a special focus on room layout, custom-designed air conditioning and vibration, which can also be adapted to proteomics, genomics, and flow or mass cytometry SRLs.

Evaluating the effectiveness of Uganda's Supranational TB Reference Laboratory quality management system training program.

BACKGROUND: Achieving the targeted organizational goals through effective training can increase employee satisfaction. Since 2015, the Supranational Reference Laboratory Uganda (SRL Uganda) has trained National Tuberculosis Reference Laboratories (NTRLs) from 21 countries in a variety of areas that cover both technical and programmatic aspects pertinent to TB laboratories. The Laboratory Quality Management System (LQMS) under SRL coordinates actions intended to ensure sustained quality of the laboratory services offered by the National TB Reference Laboratories. In order for laboratory results to be helpful in a clinical or public health setting, they must be accurate, reliable, and timely. The LQMS course aims to provide learners with knowledge on how to attain and maintain this quality. Prior to this study, there was hardly any data available on the effectiveness of LQMS trainings provided by SRL Uganda; using Kirkpatrick model, which is popular among researchers for evaluating the efficacy of the training program, this paper seeks to establish the effectiveness of the LQMS training offered by the SRL Uganda. METHOD: We evaluated the effectiveness of LQMS training within the Uganda's SRL network for courses offered during the period 2017 and 2021 for participants from the Southern and East African sub-Saharan region. RESULTS: In 2017 and 2021, respectively, test results from 10/17 and 9/17 showed overall post-test scores above 80%. Of the 18 labs evaluated, 14 showed improvement; of these, 7 labs were from the Eastern region and the other 7 were from Southern Africa; one facility in this region also maintained its accreditation. In the post-evaluation assessment, attendees of the LQMS course gave feedback of strongly agree and agree variety. CONCLUSION: More SRL Uganda network laboratories in the regions achieved a 5-star SLIPTA level rating and among these, 5 NTRLs got ISO 15189:2012 accredited by the end of 2021, while one maintained its accreditation status. This proves that the Laboratory Quality Management System training program was an effective tool in improving the quality of laboratory services, work practices, and processes.

Trait Emotional Intelligence and Self-regulated Learning in University Students during the COVID-19 pandemic: the mediation role of Intolerance of Uncertainty and COVID-19 Perceived Stress.

The coronavirus pandemic strongly impacted the higher education system, challenging university students, who were required to make a considerable effort in terms of revising their personal study approach and managing their emotions. The present study aimed to examine the impact of Trait Emotional Intelligence (TEI) on Self-Regulated Learning (SRL) strategies directly, and indirectly through the mediation of COVID-19 perceived stress (PS) and Intolerance of Uncertainty (IU). 1055 Italian university students (mean age = 22.44 years, SD = 2.39) reached via social media, filled out the tools assessing TEI, COVID-19 PS, IU and SRL. Five mediation analyses performed with PROCESS showed that TEI positively predicted all SRL dimensions, and negatively influenced COVID-19 PS and Prospective and Inhibitory IU. COVID-19 PS and Inhibitory and Prospective IU differently predicted specific SRL dimensions. The indirect effects observed suggest that TEI may help university students to cope with stressful and uncertain psychological experiences in order to achieve higher levels of SRL strategies. The study highlights that TEI plays a pivotal role in academic SRL strategies, underlining the importance of its buffering effect in distressing circumstances such as the COVID-19 pandemic.

miR-383-5p, miR-181a-5p, and miR-181b-5p as Predictors of Response to First-Generation Somatostatin Receptor Ligands in Acromegaly.

Acromegaly is a chronic systemic disease caused in the vast majority of cases by growth hormone (GH)-secreting adenoma, with surgery being the first-line treatment. When a cure is not attained with surgery, first-generation somatostatin receptor ligands (fg-SRLs) are the most common medication prescribed. Predictors of response to fg-SRLs have been studied; however, they cannot fully predict the response to fg-SRL. MicroRNAs are small RNAs, the main role of which is messenger RNA (mRNA) post-transcriptional regulation. This study aimed to identify the microRNAs involved in resistance to treatment with fg-SRLs in acromegaly. Ten patients with acromegaly undergoing treatment with fg-SRLs were selected to undergo miRNA sequencing: five controlled and five uncontrolled with treatment. Bioinformatic analysis was performed to detect differentially expressed miRNAs. Then, the same 10 samples were used for validation by qPCR and an additional 22 samples were analyzed, totaling 32 samples. e We found 59 differentially expressed miRNAs in the first analysis. miR-181a-5p and miR-181b-5p were downregulated, and miR-383-5p was upregulated in the uncontrolled group. Receiver operating characteristic (ROC) curve analysis of miR-383-5p showed an NPV of 84.3% and a PPV of 84.5%. In summary, miR-181a-5p, miR-181b-5p, and miR-383-5p are biomarkers of response to fg-SRLs, and they can be used individually or included in prediction models as tools to guide clinical decisions.

Improving Rice Leaf Shape Using CRISPR/Cas9-Mediated Genome Editing of SRL1 and Characterizing Its Regulatory Network Involved in Leaf Rolling through Transcriptome Analysis.

Leaf rolling is a crucial agronomic trait to consider in rice (Oryza sativa L.) breeding as it keeps the leaves upright, reducing interleaf shading and improving photosynthetic efficiency. The SEMI-ROLLED LEAF 1 (SRL1) gene plays a key role in regulating leaf rolling, as it encodes a glycosylphosphatidylinositol-anchored protein located on the plasma membrane. In this study, we used CRISPR/Cas9 to target the second and third exons of the SRL1 gene in the indica rice line GXU103, which resulted in the generation of 14 T0 transgenic plants with a double-target mutation rate of 21.4%. After screening 120 T1 generation plants, we identified 26 T-DNA-free homozygous double-target mutation plants. We designated the resulting SRL1 homozygous double-target knockout as srl1-103. This line exhibited defects in leaf development, leaf rolling in the mature upright leaves, and a compact nature of the fully grown plants. Compared with the wild type (WT), the T2 generation of srl1-103 varied in two key aspects: the width of flag leaf (12.6% reduction compared with WT) and the leaf rolling index (48.77% increase compared with WT). In order to gain a deeper understanding of the involvement of SRL1 in the regulatory network associated with rice leaf development, we performed a transcriptome analysis for the T2 generation of srl1-103. A comparison of srl1-103 with WT revealed 459 differentially expressed genes (DEGs), including 388 upregulated genes and 71 downregulated genes. In terms of the function of the DEGs, there seemed to be a significant enrichment of genes associated with cell wall synthesis (LOC_Os08g01670, LOC_Os05g46510, LOC_Os04g51450, LOC_Os10g28080, LOC_Os04g39814, LOC_Os01g71474, LOC_Os01g71350, and LOC_Os11g47600) and vacuole-related genes (LOC_Os09g23300), which may partially explain the increased leaf rolling in srl1-103. Furthermore, the significant downregulation of BAHD acyltransferase-like protein gene (LOC_Os08g44840) could be the main reason for the decreased leaf angle and the compact nature of the mutant plants. In summary, this study successfully elucidated the gene regulatory network in which SRL1 participates, providing theoretical support for targeting this gene in rice breeding programs to promote variety improvement.

Establishing a biosafety plan for a flow cytometry shared resource laboratory.

A biosafety plan is essential to establish appropriate practices for biosafety in a shared resource laboratory (SRL). A biosafety plan will contain the essential information for the use of biological samples on specific instrumentation, their apparent risks, and the steps that should be taken to mitigate these risks. Establishment of a biosafety plan can be a daunting task as the variety of pathogens that come through the SRL is highly diverse and may change over time; however, having a plan that can adapt to this variety will provide a framework for addressing concerns and educating personnel and users on biosafety practices. Using resources available at your institution and developing a robust relationship with health and safety personnel at your institution is key to generating an effective biosafety plan. Here we provide a basic underlying structure for a biosafety plan to aid SRL personnel in generating or maintaining their biosafety procedures, and provide guidance for establishing a dynamic, living biosafety plan.

Critical Steps in Data Management During a Crisis.

Data management is essential in a flow cytometry (FCM) shared resource laboratory (SRL) for the integrity of collected data and its long-term preservation, as described in the Cytometry publication from 2016, ISAC Flow Cytometry Shared Resource Laboratory (SRL) Best Practices (Barsky et al.: Cytometry Part A 89A(2016): 1017-1030). The SARS-CoV-2 pandemic introduced an array of challenges in the operation of SRLs. The subsequent laboratory shutdowns and access restrictions brought to the forefront well-established practices that withstood the impact of a sudden change in operations and illuminated areas that need improvement. The most significant challenges from a data management perspective were data access for remote analysis and workstation management. Notably, lessons learned from this challenge emphasize the importance of safeguarding collected data from loss in various emergencies such as fire or natural disasters where the physical hardware storing data could be directly affected. Here, we describe two data management systems that have been successful during the current emergency created by the pandemic, specifically remote access and automated data transfer. We will discuss other situations that could arise and lead to data loss or challenges in interpreting data. © 2020 International Society for Advancement of Cytometry.

Adapting to the Coronavirus Pandemic: Building and Incorporating a Diagnostic Pipeline in a Shared Resource Laboratory.

In March 2020, with lockdown due to the coronavirus pandemic underway, the Francis Crick Institute (the Crick) regeared its research laboratories into clinical testing facilities. Two pipelines were established, one for polymerase chain reaction and the other for Serology. This article discusses the Cricks Flow Cytometry Science Technology Platform (Flow STP) role in setting up the Serology pipeline. Pipeline here referring to the overarching processes in place to facilitate the receipt of human sera through to a SARs-CoV-2 enzyme-linked immunosorbent assay result. We examine the challenges that had to be overcome by a research laboratory to incorporate clinical diagnostics and the processes by which this was achieved. It describes the governance required to run the service, the design of the standard operating procedures (SOPs) and pipeline, the setting up of the assay, the validation required to show the robustness of the pipeline and reporting the results of the assay. Finally, as the lockdown started to ease in June 2020, it examines how this new service affects the daily running of the Flow STP. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Risk awareness during operation of analytical flow cytometers and implications throughout the COVID-19 pandemic.

The COVID-19 pandemic has brought biosafety to the forefront of many life sciences. The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs). In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument "failure modes." Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure. Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk. To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing. We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in "failure modes." These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated.

Biosafety during a pandemic: shared resource laboratories rise to the challenge.

Biosafety has always been an important aspect of daily work in any research institution, particularly for cytometry Shared Resources Laboratories (SRLs). SRLs are common-use spaces that facilitate the sharing of knowledge, expertise, and ideas. This sharing inescapably involves contact and interaction of all those within this working environment on a daily basis. The current pandemic caused by SARS-CoV-2 has prompted the re-evaluation of many policies governing the operations of SRLs. Here we identify and review the unique challenges SRLs face in maintaining biosafety standards, highlighting the potential risks associated with not only cytometry instrumentation and samples, but also the people working with them. We propose possible solutions to safety issues raised by the COVID-19 pandemic and provide tools for facilities to adapt to evolving guidelines and future challenges.

Real-life analysis of 280 patients with surgically treated acromegaly: a single-center experience from 2008 to 2015.

OBJECTIVE: The purpose of this study was to analyze the clinical and biochemical outcome of consecutive patients with acromegaly after microscopic transsphenoidal surgery (MTS) at a single center over an 8-year period. METHODS: A retrospective analysis of patients with acromegaly treated via MTS between 2008 and 2015 at the authors' center was performed. The mean follow-up was 29 months (range 1-120 months). Parameters investigated included tumor size, pre- and postoperative insulin-like growth factor-I, growth hormone levels, pretreatment, perioperative complications, and clinical outcome. RESULTS: A total of 280 patients with acromegaly were treated surgically at the authors' center over the abovementioned time frame and were included in analyses. For 231 of these patients, complete follow-up data were available for evaluation. One hundred eighty-eight patients (81%) showed remission initially according to current criteria. So far, 23 of these patients relapsed in the further course, so that on follow-up 165 patients (71%) demonstrated full remission by surgery alone. Most patients in whom remission after surgery failed were treated with somatostatin receptor ligands and/or dopamine agonists as second-line treatment. The main postoperative complications included transient hyponatremia and diabetes insipidus (13/280; 4.6%). CSF leakage only occurred in 2 cases (2/280; 0.7%). No surgery-related death occurred. CONCLUSIONS: The data underline the effectiveness of MTS in acromegaly. Many patients with recurrent disease or incomplete tumor resection can be successfully managed pharmacologically.

Impact of tumor characteristics and pre- and postoperative hormone levels on hormonal remission following endoscopic transsphenoidal surgery in patients with acromegaly.

OBJECTIVE: Acromegaly is a disease of acral enlargement and elevated serum levels of insulin-like growth factor-1 (IGF-1) and growth hormone (GH), usually caused by a pituitary adenoma. A lack of consensus on factors that reliably predict outcomes in acromegalic patients following endoscopic endonasal transsphenoidal surgery (EETS) warrants additional investigation. METHODS: The authors identified 52 patients with acromegaly who underwent an endoscopic endonasal approach (EEA) for resection of a GH-secreting pituitary adenoma. Preoperative and postoperative tumor and endocrinological characteristics such as tumor size, invasiveness, and GH/IGF-1 levels were evaluated as potential indicators of postoperative hormonal remission. Endocrinological remission was defined as postoperative IGF-1 levels at or below the age- and sex-normalized values. RESULTS: The 52 patients had a mean age of 50.7 ± 13.4 years and a mean follow-up duration of 24.4 ± 19.1 months. Ten patients (19%) had microadenomas and 42 (81%) had macroadenomas. Five patients (9.6%) had giant adenomas. Forty-four tumors (85%) had extrasellar extension, with 40 (77%) exhibiting infrasellar invasion, 18 (35%) extending above the sella, and 7 (13%) invading the cavernous sinuses. Thirty-six patients (69%) underwent gross-total resection (GTR; mean maximal tumor diameter 1.47 cm), and 16 (31%) underwent subtotal resection (STR; mean maximal tumor diameter 2.74 cm). Invasive tumors were significantly larger, and Knosp scores were negatively correlated with GTR. Thirty-eight patients (73%) achieved hormonal remission after EEA resection alone, which increased to 87% with adjunctive medical therapy. Ninety percent of patients with microadenomas and 86% of patients with macroadenomas achieved hormonal remission. Preoperative IGF-1 and postoperative day 1 (POD1) GH levels were inversely correlated with hormonal remission. Postoperative CSF leakage occurred in 2 patients (4%), and none experienced vision loss, death, or injury to internal carotid arteries or cranial nerves. CONCLUSIONS: Endoscopic transsphenoidal resection of GH-secreting pituitary adenomas is a safe and highly effective treatment for achieving hormonal remission and tumor control in up to 87% of patients with acromegaly when combined with postoperative medical therapy. Patients with lower preoperative IGF-1 and POD1 GH levels, with less invasive pituitary adenomas, and who undergo GTR are more likely to achieve postoperative biochemical remission.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

Automating the Shared Resource Laboratory Using Computer Scripts: A Case Report.

Shared resource laboratories (SRLs) offer instrumentation, training, and support to investigators and play an important role in the progress and development of science. To facilitate daily tasks and to provide an effective service, we have made use of computer scripts; a list of computer commands that are processed sequentially, to automate tasks in our flow cytometry facility. Using Python and an application programming interface (API), we automate user communication and produce a daily schedule display screen. We exploit the accessible nature of open standards to use R and Python to analyze and backup data from the BD Influx cell sorter. Finally, we show that through simple scripting, we can add value to an existing service by producing sort statistics from the Beckman Coulter XDP cell sorter. With these five examples, we demonstrate and wish to inspire other SRLs that the use of scripts helps to improve work efficiency, can solve problems, and can enhance the service provided by the SRL. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Patterns in spatial distribution and root trait syndromes for ecto and arbuscular mycorrhizal temperate trees in a mixed broadleaf forest.

Functional differences between trees with arbuscular (AM) or ectomycorrhizal (ECM) partnerships influence important ecological processes including nutrient cycling, community assembly, and biomass allocation patterns. Although most broadleaf temperate forests show both mycorrhizal types, relatively few studies have addressed functional difference among coexisting mycorrhizal tree species. The maintenance of ECM associations usually requires higher C investment than AM, leading to (A) lower root biomass and (B) more conservative root trait syndromes in ECM tree species compared to AM species. Here we quantified the representation and trait syndromes of 14 canopy tree species associated with either AM or ECM fungi in a natural forest community. Our results showed that, whereas species root abundance was proportional to basal area, some ECM tree roots were largely under-represented (up to ~ 33%). Most of the under-representation was due to lower than expected root abundance of Quercus rubra and Fagus grandifolia. Functional root traits in tree species were similar, with the exception of higher tissue density in ECM species. Moreover, closely related AM and ECM exhibited similar traits, suggesting inherited trait syndrome from a common ancestor. Thus, we found little evidence of divergent functional root trait syndromes between mycorrhizal types. Cores dominated by ECM species influenced trait distribution at the community level, but not total biomass, suggesting that mycorrhizal affiliation may have a stronger effect on the spatial distribution of traits but not on biomass stocks. Our results present an important step toward relating belowground carbon dynamics to species traits, including mycorrhizal type, in broadleaf temperate forests.

A worldview of root traits: the influence of ancestry, growth form, climate and mycorrhizal association on the functional trait variation of fine-root tissues in seed plants.

Fine-root traits play key roles in ecosystem processes, but the drivers of fine-root trait diversity remain poorly understood. The plant economic spectrum (PES) hypothesis predicts that leaf and root traits evolved in coordination. Mycorrhizal association type, plant growth form and climate may also affect root traits. However, the extent to which these controls are confounded with phylogenetic structuring remains unclear. Here we compiled information about root and leaf traits for > 600 species. Using phylogenetic relatedness, climatic ranges, growth form and mycorrhizal associations, we quantified the importance of these factors in the global distribution of fine-root traits. Phylogenetic structuring accounts for most of the variation for all traits excepting root tissue density, with root diameter and nitrogen concentration showing the strongest phylogenetic signal and specific root length showing intermediate values. Climate was the second most important factor, whereas mycorrhizal type had little effect. Substantial trait coordination occurred between leaves and roots, but the strength varied between growth forms and clades. Our analyses provide evidence that the integration of roots and leaves in the PES requires better accounting of the variation in traits across phylogenetic clades. Inclusion of phylogenetic information provides a powerful framework for predictions of belowground functional traits at global scales.

Root traits are more than analogues of leaf traits: the case for diaspore mass.

Root traits are often thought to be analogues of leaf traits along the plant economics spectrum. But evolutionary pressures have most likely shaped above- and belowground patterns differentially. Here, we aimed to identify the most important aboveground traits for explaining root traits without an a priori focus on known concepts. We measured morphological root traits in a glasshouse experiment on 141 common Central European grassland species. Using random forest algorithms, we built predictive models of six root traits from 97 aboveground morphological, ecological and life history traits. Root tissue density was best predicted by leaf dry matter content, whereas traits related to root fineness were best predicted by diaspore mass: the heavier the diaspore, the coarser the root system. Specific leaf area (SLA) was not an important predictor for any of the root traits. This study confirms the hypothesis that root traits are more than analogues of leaf traits within a plant economics spectrum. The results reveal a novel ecological pattern and highlight the power of root data to close important knowledge gaps in trait-based ecology.

Pegvisomant in acromegaly: an update.

BACKGROUND: In 2007, we published an opinion document to review the role of pegvisomant (PEG) in the treatment of acromegaly. Since then, new evidence emerged on the biochemical and clinical effects of PEG and on its long-term efficacy and safety. AIM: We here reviewed the emerging aspects of the use of PEG in clinical practice in the light of the most recent literature. RESULTS: The clinical use of PEG is still suboptimal, considering that it remains the most powerful tool to control IGF-I in acromegaly allowing to obtain, with a pharmacological treatment, the most important clinical effects in terms of signs and symptoms, quality of life and comorbidities. The number of patients with acromegaly exposed to PEG worldwide has become quite elevated and the prolonged follow-up allows now to deal quite satisfactorily with many clinical issues including major safety issues, such as the concerns about possible tumour (re)growth under PEG. The positive or neutral impact of PEG on glucose metabolism has been highlighted, and the clinical experience, although limited, with sleep apnoea and pregnancy has been reviewed. Finally, the current concept of somatostatin receptor ligands (SRL) resistance has been addressed, in order to better define the acromegaly patients to whom the PEG option may be offered. CONCLUSIONS: PEG increasingly appears to be an effective and safe medical option for many patients not controlled by SRL but its use still needs to be optimized.