Patient-reported outcome measures for primary hyperparathyroidism: a systematic review of measurement properties.

BACKGROUND: The quality of patient-reported outcome measures (PROMs) used to assess the outcomes of primary hyperparathyroidism (PHPT), a common endocrine disorder that can negatively affect patients' health-related quality of life due to chronic symptoms, has not been rigorously examined. This systematic review aimed to summarize and evaluate evidence on the measurement properties of PROMs used in adult patients with PHPT, and to provide recommendations for appropriate measure selection. METHODS: After PROSPERO registration (CRD42023438287), Medline, EMBASE, CINAHL Complete, Web of Science, PsycINFO, and Cochrane Trials were searched for full-text articles in English investigating PROM development, pilot studies, or evaluation of at least one PROM measurement property in adult patients with any clinical form of PHPT. Two reviewers independently identified studies for inclusion and conducted the review following the Consensus-Based Standards for the Selection of Health Measurement Instruments (COSMIN) Methodology to assess risk of bias, evaluate the quality of measurement properties, and grade the certainty of evidence. RESULTS: From 4989 records, nine PROM development or validation studies were identified for three PROMs: the SF-36, PAS, and PHPQoL. Though the PAS demonstrated sufficient test-retest reliability and convergent validity, and the PHPQoL sufficient test-retest reliability, convergent validity, and responsiveness, the certainty of evidence was low-to-very low due to risk of bias. All three PROMs lacked sufficient evidence for content validity in patients with PHPT. CONCLUSIONS: Based upon the available evidence, the SF-36, PAS, and PHPQoL cannot currently be recommended for use in research or clinical care, raising important questions about the conclusions of studies using these PROMs. Further validation studies or the development of more relevant PROMs with strong measurement properties for this patient population are needed.

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture.

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.

Use of patient-reported outcome measures after breast reconstruction in low- and middle-income countries: a scoping review.

BACKGROUND: Patient-reported outcome measures (PROMs) are increasingly administered in high-income countries to monitor health-related quality of life of breast cancer patients undergoing breast reconstruction. Although low- and middle-income countries (LMICs) face a disproportionate burden of breast cancer, little is known about the use of PROMs in LMICs. This scoping review aims to examine the use of PROMs after post-mastectomy breast reconstruction among patients with breast cancer in LMICs. METHODS: MEDLINE, Embase, Web of Science, CINAHL, and PsycINFO were searched in August 2022 for English-language studies using PROMs after breast reconstruction among patients with breast cancer in LMICs. Study screening and data extraction were completed. Data were analyzed descriptively. RESULTS: The search produced 1024 unique studies, 33 of which met inclusion criteria. Most were observational (48.5%) or retrospective (33.3%) studies. Studies were conducted in only 10 LMICs, with 60.5% in China and Brazil and none in low-income countries. Most were conducted in urban settings (84.8%) and outpatient clinics (57.6%), with 63.6% incorporating breast-specific PROMs and 33.3% including breast reconstruction-specific PROMs. Less than half (45.5%) used PROMs explicitly validated for their populations of interest. Only 21.2% reported PROM response rates, ranging from 43.1 to 96.9%. Barriers and facilitators of PROM use were infrequently noted. CONCLUSIONS: Despite the importance of PROM collection and use in providing patient-centered care, it continues to be limited in middle-income countries and is not evident in low-income countries after breast reconstruction. Further research is necessary to determine effective methods to address the challenges of PROM use in LMICs.

Feasibility of implementing patient-reported outcome measures into routine breast cancer care delivery using a novel collection and reporting platform.

Objectives: imPROVE is a new Health Information Technology platform that enables systematic patient-reported outcome measure (PROM) collection through a mobile phone application. The purpose of this study is to describe our initial experience and approach to implementing imPROVE among breast cancer patients treated in breast and plastic surgery clinics. Materials and Methods: We describe our initial implementation in 4 phases between June 2021 and February 2022: preimplementation, followed by 3 consecutive implementation periods (P1, P2, P3). The Standards for Reporting Implementation Studies statement guided this study. Iterative Plan-Do-Study-Act (PDSA) cycles supported implementation, and success was evaluated using the Reach, Effectiveness, Adoption, Implementation, and Maintenance framework. Results: Qualitative interviews conducted during the preimplementation phase elicited 4 perceived implementation barriers. Further feedback collected during each phase of implementation resulted in the development of brochures, posters in clinic spaces, and scripts for clinic staff to streamline discussions with patients, and the resolution of technical issues concerning patient login capabilities, such as compatibility with cell phone software and barriers to downloading imPROVE. Feedback also generated ideas for facilitating provider interpretation of PROM results. By the end of P3, 2961 patients were eligible, 1375 (46.4%) downloaded imPROVE, and 1070 (36.1% of those eligible, 78% of those who downloaded) completed at least 1 PROM. Discussion and Conclusion: Implementation efforts across 2 surgical departments at 2 academic teaching hospitals enabled collaboration across clinical specialties and longitudinal PROM reporting for patients receiving breast cancer care; the implementation effort also highlighted patient difficulties with mobile app-based PROM collection, particularly around initial engagement.

Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Modulation of nutrient precursors for controlling metabolic inhibitors by genome-scale flux balance analysis.

Therapeutic protein productivity and glycosylation pattern highly rely on cell metabolism. Cell culture medium composition and feeding strategy are critical to regulate cell metabolism. In this study, the relationship between toxic metabolic inhibitors and their nutrient precursors was explored to identify the critical medium components toward cell growth and generation of metabolic by-products. Generic CHO metabolic model was tailored and integrated with CHO fed-batch metabolomic data to obtain a cell line- and process-specific model. Flux balance analysis study was conducted on toxic metabolites cytidine monophosphate, guanosine monophosphate and n-acetylputrescine-all of which were previously reported to generate from endogenous cell metabolism-by mapping them to a compartmentalized carbon utilization network. Using this approach, the study projected high level of inhibitory metabolites accumulation when comparing three industrially relevant fed-batch feeding conditions one against another, from which the results were validated via a dose-dependent amino acids spiking study. In the end, a medium optimization design was employed to lower the amount of supplemented nutrients, of which improvements in critical process performance were realized at 40% increase in peak viable cell density (VCD), 15% increase in integral VCD, and 37% increase in growth rate. Tight control of toxic by-products was also achieved, as the study measured decreased inhibitory metabolites accumulation across all conditions. Overall, the study successfully presented a digital twin approach to investigate the intertwined relationship between supplemented medium constituents and downstream toxic metabolites generated through host cell metabolism, further elucidating different control strategies capable of improving cellular phenotypes and regulating toxic inhibitors.

Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture.

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.

Insight into the substrate length restriction of M32 carboxypeptidases: characterization of two distinct subfamilies.

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism.

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.

Overexpression of barley hva1 gene in creeping bentgrass for improving drought tolerance.

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T(0) generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T(0) transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.

Behçet disease: recommendation for clinical management of mucocutaneous lesions.

Mucocutaneous lesions are the most common manifestation of Behçet disease. These lesions can often become refractory to multiple treatments and present challenges to physicians. In this article, different treatments for mucocutaneous lesions in Behçet disease are reviewed and discussed. Topical or intralesional corticosteroids, oral pentoxifylline, sucralfate, dapsone, colchicine, and systemic low-dose corticosteroids, used either alone or in combination, are safe and having varying evidence for effect in mild to moderate mucocutaneous disease. Azathioprine or methotrexate can be used if the lesions are refractory to the previously mentioned therapies. Tumor necrosis factor (TNF) inhibitors such as infliximab or etanercept should be considered as the next step in the treatment if azathioprine or methotrexate fails. Tacrolimus, cyclosporine, and interferon-alpha-2a should be used generally only if TNF inhibitors have failed as a result of their toxicities.

Current tumor necrosis factor-alpha inhibitor use is associated with a higher probability of remissions in patients with rheumatoid arthritis.

OBJECTIVE: To determine if current tumor necrosis factor-alpha (TNF-alpha) inhibitor use is associated with a higher probability of remission than non-use in patients with rheumatoid arthritis (RA). METHODS: Clinical and demographic data were collected from 322 patients with RA during regularly scheduled clinic visits. Current and past medications were recorded. Disease activity status (remission or not) was determined using American College of Rheumatology preliminary criteria for clinical remission of RA. A logistic regression analysis was used to calculate crude and adjusted odds ratios (OR) for remission for current TNF-alpha inhibitor users versus non-users. Multivariate analysis included age, gender, race, disease duration, use of nonsteroidal antiinflammatory drugs (NSAID), prednisone dosage, and numbers of previously used disease modifying antirheumatic drugs (DMARD). RESULTS: Of the 111 patients enrolled in the study who were users of TNF-alpha inhibitors, 25.2% were found to be in clinical remission. Of the 211 patients who were non-users, 14.7% were in clinical remission. The unadjusted OR for remission in TNF-alpha inhibitor users was 1.96 (95% confidence interval, CI: 1.10 to 3.48). The adjusted OR was 2.74 (95% CI: 1.40 to 5.34). CONCLUSION: Cross-sectional observations from an outpatient arthritis clinic found a significantly higher remission rate in patients with RA taking a TNF-alpha inhibitor compared to non-users.

In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass.

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.

Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system.

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression.

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

Linkage of AFLP markers to lhd 1, a recessive heterochronic gene in Italian ryegrass.

A mutant, leafy head I (lhd 1), was discovered in Japan from the progeny of Italian ryegrass (Lolium multiflorum Lam.) 'Nioudachi' and local line 'Aichikei #3'. Compared with normal plants, the mutant plant is a dwarf with a larger number of intenodes per stem, shorter internodes, and smaller leaves. The plants also head later in the season. Aerial roots are usually produced from the stem nodes during rainy seasons. In characterizing lhd 1, it was found to have many branches with small leaves and many small panicles on the upper part of the plant. Panicle development was severely disturbed in lhd 1 mutants, and the number of leaves produced in the vegetative phase was nearly twice that produced in the wild-type counterpart. The lhd 1 mutant appears to be a heterochronic mutation that is able to extend the vegetative period during development. The frequency of mutants in segregating populations indicated that lhd 1 is a recessive allele. To determine the linkage relationship between the lhd 1 gene and AFLP markers,768 primer combinations were screened for polymorphisms using bulked segregant analyses in two populations with 316 and 30 plants, respectively. Five AFLP markers were linked to the lhd 1 locus. E3/M41-1 and E16/M14-2 cosegregated with lhd 1. E16/M14-1 and E30/M10-1 flanked the gene at a distance of 0.3 cM and E30/M14-2 was linked to lhd 1 at a distance of 0.6 cM.