Extreme Symptom Burden for Patients With COVID-19 at the End of Life; Extrapolation of Knowledge Gained to Achieve Sustained Comfort and Dignity for all Patients in Their Last Days of Life1.

BACKGROUND: We describe two complex cases in the setting of COVID-19 at the End of Life, to enhance learning for all patients. CASE PRESENTATION: Maintenance of sustained comfort in two cases required multiple drugs, specifically selected for symptoms that necessitated three separate pumps delivering continuous 24-hour subcutaneous infusion. CASE MANAGEMENT: Management of sustained comfort included opioid, midazolam, anti-secretory, diclofenac for intractable temperature, phenobarbital for extreme agitation, in one, where seizure activity was present, while insomnia, was a prominent feature of another. Management of Akatasia was also required. CASE OUTCOME: Attention to each individual patient's rapidly evolving symptoms, during the dying phase, with a thorough differential diagnosis, wa s vitally important in the context of a 'Good Death'. This was achieved in both cases, reflected by evidence at the bedside of comfort and a minimum need for 'as required' drugs in the last days of life. CONCLUSIONS: COVID-19 being a new illness, we need to prospectively study the symptom burden/clustering at End of Life and learn from management of this new disease for other illnesses also. Further research is required to develop protocols on; when does Midazolam dose reach tolerance and when should an alternative drug such as phenobarbital for sustained Gamma-Aminobutyric Acid effects be initiated; examine the optimal approach to sustained temperature control; be cognisant of extrapyramidal side effects of drugs used at End of Life and consider looking at a lack of need for 'as required' drugs in the last days of life as an outcome measure of sustained comfort.

Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.881C > T; p.Ala294Val (NUIGi059-A, NUIGi059-B, NUIGi059-C) and 3 healthy controls (NUIGi060-A, NUIGi060-B, NUIGi060-C).

Developmental and epileptic encephalopathies (DEEs) are a group of severe, early-onset epilepsies which are often caused by genetic mutations in ion channels. Mutations in KCNQ2, which encodes the voltage-gated potassium channel Kv7.2, is known to cause DEE. Here, we generated three iPSC lines from dermal fibroblasts of a 5 year-old male patient with the KCNQ2 c.881C > T (p.Ala294Val) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines have been validated by SNP karyotyping, STR analysis, expression of pluripotent genes, the capacity to differentiate into three germ layers and confirmation of the mutation in the patient.

On the Influence of Heat Input on Ni-WC GMAW Hardfaced Coating Properties.

Hardfacing is one of the techniques used for part lifecycle elongation. Despite being used for over 100 years, there still is much to discover, as modern metallurgy provides more and more sophisticated alloys, which then have to be studied to find the best technological parameters in order to fully utilize complex material properties. One of the most efficient and versatile hardfacing approaches is Gas Metal Arc Welding technology (GMAW) and its cored-wire equivalent, known as FCAW (Flux-Cored/Cored Arc Welding). In this paper, the authors study the influence of heat input on the geometrical properties and hardness of stringer weld beads fabricated from cored wire consisting of macrocrystalline tungsten carbides in a nickel matrix. The aim is to establish a set of parameters which allow to manufacture wear-resistant overlays with high deposition rates, preserving all possible benefits of this heterogenic material. This study shows, that for a given diameter of the Ni-WC wire, there exists an upper limit of heat input beyond which the tungsten carbide crystals may exhibit undesired segregation at the root.

Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.638C > T; p.Arg213Gln (NUIGi063-A, NUIGi063-B, NUIGi063-C) and 3 healthy controls (NUIGi064-A, NUIGi064-B, NUIGi064-C).

KCNQ2 encodes the potassium-gated voltage channel Kv7.2, responsible for the M-current, which contributes to neuronal resting membrane potential. Pathogenic variants in KCNQ2 cause early onset epilepsies, developmental and epileptic encephalopathies. In this study, we generated three iPSC lines from dermal fibroblasts of a 5 year-old female patient with the KCNQ2 c.638C > T (p.Arg213Gln) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines were validated by confirming the targeted mutation, SNP karyotyping, STR analysis, pluripotent gene expression, differentiation capacity into three germ layers, and were free of transgene integration and Mycoplasma.

Tool Wear Issues in Hot Forging of Steel.

Steel forging tools are subjected to a number of tribological wear mechanisms depending on the geometry and surface of the tool and the flow of the material. Thus, there is no single general tribological wear mechanism, and only the predominant wear mechanisms in this case can be indicated. The problem has been known for years, but due to its complexity research on it is still relevant. In this study, the various wear mechanisms of hot work tools are analyzed on the basis of original research. Moreover, the influence of the micro- and macrostructure of the material and of its mechanical, physical, and technological characteristics on susceptibility to a given type of wear is considered. Adhesive wear, wear caused by plastic deformation, mechanical fatigue, thermal fatigue, the influence of hardness, heat treatment, and impact strength on tool wear and the mechanisms causing this wear are discussed in addition to tribological wear mechanisms such as abrasive wear. The influence of thermomechanical history and the characteristics of the tool material, including structural anisotropy, on the wear of these tools is indicated. The analysis of wear mechanisms performed will enable more precise definition of the principles of tool material selection and tool material condition for the hot forging of steel.

The miR-17-92 cluster: Yin and Yang in human cancers.

MicroRNAs (miRNAs) are non-coding RNAs which modulate gene expression via multiple post-transcriptional mechanisms. They are involved in a variety of biological processes, including cell proliferation, metastasis, metabolism, tumorigenesis, and apoptosis. Dysregulation of miRNA expression has been implicated in human cancers, and they may also serve as biomarkers of disease progression and prognosis. The miR-17-92 cluster is one of the most widely studied miRNA clusters, which was initially reported as an oncogene, but was later reported to exhibit tumour suppressive effects in some human cancers. This review summarizes the recent progress and context-dependant role of this cluster in various cancers. We summarize the known mechanisms which regulate miR-17-92 expression and molecular pathways that are in turn controlled by it. We discuss examples where it acts as an oncogene or a tumour suppressor along with key targets affecting hallmarks of cancer. We discuss how cellular contexts regulate the biological effects of miR-17-92. The plausible mechanisms of its paradoxical roles are explained, and mechanisms are described that may contribute to cell fate regulation by miR-17-92. Further, we discuss recently developed strategies to target miR-17-92 cluster in human cancers. MiR-17-92 may serve as a potential biomarker for prognosis and response to therapy as well as a target for cancer prevention and therapeutics.

Hot Deformation Behavior of 4130 High-Strength Steel.

Hot deformation behavior of 4130 steel and optimization of its processing parameters are presented in this paper. Compression tests were performed at temperatures ranging from 800 to 1200 °C and at the strain rates in the range from 0.01 to 100 s-1. A comprehensive analysis of the material behavior at different temperature and strain-rate ranges was performed taking into account various criteria of stability and instability of the material flow under various thermomechanical conditions. The flow-stress curves obtained during compression tests, as well as the processing maps elaborated on the basis of various flow-stability criteria, are discussed. Processing parameters developed according to the Prasad's and Murty's criteria are recommended for designing the technology of forging of the investigated steel. Such parameters ensure the homogeneity and stability of the material flow in a forged part, what was confirmed by successful forging of 4130 steel in industrial conditions. The processing map developed according to Gegel's approach, as compared to the processing maps obtained in accordance with the Prasad's and Murty's criteria, should be treated as general support for determining the thermomechanical processing parameters.

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

Reference intervals for clinical biochemistry and haematology tests during normal pregnancy.

BACKGROUND: Pregnancy induces physiological changes which affect biochemical and haematological parameters. As the significance of laboratory test results change throughout pregnancy, the reference interval (RI) or key result interpretive guide should be specific to pregnancy. This study sought to establish trimester-specific-RIs for routine biochemical and haematological tests in healthy white European women with singleton pregnancies with comparison to RIs for non-pregnant European adults. METHODS: A retrospective analysis of a prospective longitudinal single-centre study of healthy pregnant women conducted between November 2018 and December 2020 in a tertiary academic hospital with approximately 3000 births annually. Inclusion criteria: signed informed consent, age ≥18 years, white European, body mass index (BMI) <25 kg/m2, blood pressure <140/90mmHg, non-smoker, no previous pathology or gestational diabetes. Trimester defined as T1: up to 13 weeks + 6 days, T2: 14-27 weeks + 6 days and T3: ≥28-41 weeks + 6 days. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 31 biochemical and 10 haematological ISO15189:2012 accredited tests were measured using Roche Cobas® and Sysmex XN-9100™ analysers, respectively. RIs were established according to the International Federation of Clinical Chemistry (IFCC) recommended method. RESULTS: Apparently healthy pregnant women (n = 124) with bio-banked serum samples in each trimester were recruited. At the booking visit, 49.2% (n = 61) of participants were nulliparous, with median age of 34.4 (IQR: 31.3-37.3) years, gestational age of 89 (IQR: 84-93) days, BMI of 22.5 (IQR: 21.0-23.7) kg/m2 and systolic and diastolic blood pressure of 116 (110-125) mmHg and 67 (61-75) mmHg, respectively. CONCLUSIONS: Normative trimester-specific biological intervals for routinely requested biochemical and haematological medical laboratory tests were established. These RIs will be invaluable to result interpretation and the management of pregnant women.

MiRNA as a Potential Target for Multiple Myeloma Therapy-Current Knowledge and Perspectives.

Multiple myeloma (MM) is the second most common hematological malignancy. Despite the huge therapeutic progress thanks to the introduction of novel therapies, MM remains an incurable disease. Extensive research is currently ongoing to find new options. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post-transcriptional level. Aberrant expression of miRNAs in MM is common. Depending on their role in MM development, miRNAs have been reported as oncogenes and tumor suppressors. It was demonstrated that specific miRNA alterations using miRNA mimics or antagomirs can normalize the gene regulatory network and signaling pathways in the microenvironment and MM cells. These properties make miRNAs attractive targets in anti-myeloma therapy. However, only a few miRNA-based drugs have been entered into clinical trials. In this review, we discuss the role of the miRNAs in the pathogenesis of MM, their current status in preclinical/clinical trials, and the mechanisms by which miRNAs can theoretically achieve therapeutic benefit in MM treatment.

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with "no-option" critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial.

BACKGROUND AIMS: Approximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those "no-option" patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing. METHODS: Five bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units-fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow. RESULTS: CLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors' marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL. CONCLUSIONS: In addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.

GMP-Compliant Production of Autologous Adipose-Derived Stromal Cells in the NANT 001 Closed Automated Bioreactor.

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

Phase 2 studies of lenalidomide, subcutaneous bortezomib, and dexamethasone as induction therapy in patients with newly diagnosed multiple myeloma.

There are limited prospective data on lenalidomide, subcutaneous bortezomib, and dexamethasone (RsqVd) in transplant-eligible/transplant-ineligible patients with newly diagnosed multiple myeloma. Reliable biomarkers for efficacy and toxicity are required to better tailor therapy. Two parallel studies were conducted by Cancer Trials Ireland (CTI; NCT02219178) and the Dana-Farber Cancer Institute (DFCI; NCT02441686). Patients received four 21-day cycles of RsqVd and could then receive either another 4 cycles of RsqVd or undergo autologous stem cell transplant. Postinduction/posttransplant, patients received lenalidomide maintenance, with bortezomib included for high-risk patients. The primary endpoint was overall response rate (ORR) after 4 cycles of RsqVd. Eighty-eight patients were enrolled and 84 treated across the two studies; median age was 64.7 (CTI study) and 60.0 years (DFCI study), and 59% and 57% had stage II-III disease. Pooled ORR after 4 cycles in evaluable patients was 93.5%, including 48.1% complete or very good partial responses (CTI study: 91.9%, 59.5%; DFCI study: 95.0%, 37.5%), and in the all-treated population was 85.7% (44.0%). Patients received a median of 4 (CTI study) and 8 (DFCI study) RsqVd cycles; 60% and 31% of patients (CTI study) and 33% and 51% of patients (DFCI study) underwent transplant or received further RsqVd induction, respectively. The most common toxicity was peripheral neuropathy (pooled: 68%, 7% grade 3-4; CTI study: 57%, 7%; DFCI study: 79%, 7%). Proteomics analyses indicated elevated kallikrein-6 in good versus poor responders, decreased midkine in good responders, and elevated macrophage inflammatory protein 1-alpha in patients who stopped treatment from neurotoxicity, suggesting predictive biomarkers warranting further investigation.

Case of recurrent Achromobacter xylosoxidans bacteraemia and PICC (peripherally-inserted central catheter) line infection in an immunocompromised patient.

BACKGROUND: This report describes recurrent A. xylosoxidans bloodstream and PICC (peripherally-inserted central catheter) line infection in an immunocompromised patient. PRESENTATION OF CASE: A 64-year-old female with acute promyelocytic leukaemia presented during a non-neutropenic febrile episode, and A. xylosoxidans was isolated from multiple PICC and peripheral blood cultures, and from the tip of the line on removal. The patient was treated with meropenem and a new PICC line was inserted after sterile blood cultures. Six weeks later, she represented with A. xylosoxidans from multiple cultures from the line. She was treated with piperacillin-tazobactam and the line was removed. There was no evidence of deep-seated infection. Further discussion revealed that the patient was using a sponge to clean, and a sleeve to cover her PICC-line while bathing. A. xylosoxidans was cultured from both the sponge and the swab. Whole Genome Sequencing performed on two blood culture isolated and both environmental isolates confirmed all four isolates were indistinguishable. The patient was advised not to use the sponge/sleeve in future and we have incorporated specific advice in this regard into our patient information. DISCUSSION: Achromobacter xylosoxidans is an aerobic, non-lactose fermenting gram-negative bacillus usually considered an opportunistic pathogen. It is associated with infection in immunocompromised patients, and is an emerging pathogen in catheter-related infections, sometimes associated with contaminated water. CONCLUSION: This case of recurrent A. xylosoxidans line infection highlights diagnostic and management challenges associated with catheter-related infections. Treatment is challenging because of intrinsic and acquired resistance mechanisms. Empiric treatment with anti-pseudomonal penicillins or carbapenems with line removal is typically required.

Reference intervals for commonly requested biochemical and haematological parameters in a healthy Irish adult Caucasian population.

INTRODUCTION: In laboratory medicine, reference intervals (RIs) are key decision support tools used to guide the clinical interpretation of numerical test results. Best practice suggests each laboratory establishes RIs in the local population prior to introducing an assay into routine clinical practice. AIM: The aim of this study was to define RIs for frequently requested biochemical/haematological parameters in a healthy adult Irish Caucasian population. METHODS: A cross-sectional study of non-pregnant apparently healthy volunteers was conducted. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 37 commonly requested biochemical (serum, n = 26) and haematological (venous blood, n = 11) ISO15189:2012 accredited tests were analysed, using the Roche Cobas® Sebia Capillarys 3 Tera and Siemens Advia® 2120i platforms following standard operating procedures. RIs were defined according to the International Federation of Clinical Chemistry (IFCC) recommended method. RESULTS: Of 208 apparently healthy volunteers, 76 failed to meet the study inclusion criteria. The reference population comprised of 132 participants (males: n = 65, 49.2%) with a median age of 29.7 (18.1-62.2) years. RIs for the majority of biochemical/haematological parameters were broadly in accord with those provided by Pathology Harmony (UK)/Irish RI Harmonisation Project and the manufacturer Roche Diagnostics. However, the established RI defined for HbA1c: 27-37 mmol/mol was markedly different from that quoted nationally, HbA1c: 20-42 mmol/mol. CONCLUSION: Normative biological intervals established in a healthy adult Irish population for 37 commonly requested biochemical/haematological parameters will be a valuable aid to result interpretation in clinical laboratories after appropriate verification in accordance with ISO 15189: 2012.

Wear Mechanisms of the Forging Tool Used in Pre-Forming in a Double Forging System of Truck Parts.

Tool life in plastic forming processes is a problem of the utmost importance as it significantly affects the cost of production. Hot forging with hammers and mechanical presses is an example of the technological process in which the load on tools is extremely high and, consequently, the lifetime of tools is short. Considering, additionally, that this applies to large-scale production, from an economic point of view, the key issue will be to extend the tool life, make an accurate prediction of the number of parts that can be forged before the replacement of dies is necessary, and develop a system for quick tool changeover. Initially, however, it is necessary to understand the causes of excessive tool wear, which may lie in phenomena occurring at the level of microstructure. The aim of this article was to outline an example of the coexistence of multiple wear mechanisms in hot forging dies. For the modified chemical composition, the microstructure examinations were performed in selected areas of the tool. The research has revealed the causes of cracks in tools and some irregularities in the preparation of tools for production process.

The Role of Chemical Composition of High-Manganese Cast Steels on Wear of Excavating Chain in Railway Shoulder Bed Ballast Cleaning Machine.

The main task for a ballast bed is to transmit the sleeper pressure in a form of stress cone to the subsoil, provide proper drainage and resist the sleeper displacement. Poorly maintained ballast could severely limit the maximum speed capacity and create further problems with the structural integrity, possibly leading to a complete failure of a given rail line. To prevent the unwanted corollaries, the ballast bed has to be periodically cleaned with an appropriate machinery. In this paper the authors investigated the effect of the chemical composition on the physical properties of the ballast excavating chains made of high-manganese steels. The authors focused on the wear mechanism, work hardening ability and hardness in the cross-sections areas. A microstructure analysis was performed as well, and observations revealed divergent morphology of precipitations at the grain boundaries, which influenced the size of austenite grains. The deformation twins formed as a result of operation were noticed in the samples. Research has shown that less carbon and chromium reduces the hardness of cast steel, and it specifically affects the ability to strain hardening. The authors explained the role of adjustments in chemical composition in the operational properties of high-manganese cast steels. It has been shown in the paper that different chemical compositions affect the properties of the alloys, and this causes different types of wear. The high content of chromium increases the hardness of materials before and after plastic deformation hardening, which in the conditions of selector chains results in greater dimensional stability during wear of holes in pin joints and will be more susceptible to abrasive wear in the presence of dusts from the ballast than creep.

Generation and characterization of three induced pluripotent stem cell lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) from a 51-year-old healthy individual.

Skin punch biopsy was donated by a healthy 51-year-old Caucasian male and the dermal fibroblasts were reprogrammed into human induced pluripotent stem cell (hiPSC) lines by using non-integrative Sendai viruses expressing OCT4, SOX2, KLF4 and c-MYC. Three iPSC lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) highly expressed the pluripotent markers and were capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin. These iPSCs can be offered as controls and in combination with genome-editing and three-dimensional (3D) system. They may be used for human disease modelling and drug screening.