bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and comparison of sex‐specific serious adverse drug reactions in spontaneous reports and systematically collected reports (ADRED)
Authors: Christ Patrick , Dubrall Diana , Just Katja Susanne , Lewke Britta , Below Maike , Stingl Julia Carolin , Schmid Matthias , Sachs Bernhardt
Published: October 2023

Aims

Adverse drug reactions (ADRs) are known to show sex‐specific differences in occurrence and phenotype. The aim of this study was to analyse sex‐specific differences in ADR–drug combinations that required hospitalization based on two different datasets.

Methods

We performed a complementary analysis of (i) spontaneously reported (n = 12 564, female = 51.7%) and (ii) systematically collected ADR reports from a prospective multicentre observational study (ADRED, n = 2355, female = 48.2%) from Germany in the ADR database EudraVigilance (EV). Both datasets were analysed separately concerning the suspected drugs, ADRs and ADR–drug combinations more frequently reported for females or males by calculating reporting odds ratios (ROR) with 95% confidence intervals. ADR–drug combinations more frequently reported for either females or males in EV reports were related to prescription data. Finally, the results from both datasets were discussed with regard to their (dis‐)concordance.

Results

In both datasets, some antineoplastic agents and nervous system drugs were found to be reported more often for females than males (RORs ranging from 1.5 [1.1–2.1] for quetiapine in spontaneous reports to 41.3 [13.1–130.0] for trastuzumab in spontaneous reports). ADRs of the respiratory system, and haemorrhages were described predominantly for males in both datasets. In spontaneous reports the ADR–drug combination self‐injurious behaviour–quetiapine was more often reported for females without and with consideration of drug prescriptions (ROR: 3.8 [1.3–11.0]). Quetiapine and psychiatric disorders (superordinate level) was exclusively reported for females in ADRED reports.

Conclusions

Our results can contribute to raise awareness and further knowledge regarding sex‐specific ADRs. The findings require further in‐depth investigation.
External validation of a pharmacokinetic model for target‐controlled infusion of cefazolin as a prophylactic antibiotic
Authors: Lee Sou Hyun , Yoon Hyeongseo , Park Junik , Choi Jae Moon , Kim Kyung Mi , Lee Eun‐Kyung , Noh Gyu‐Jeong , Moon Ju‐Yeon , Cho Byung‐Moon
Published: October 2023

Aims

This study aimed to evaluate the predictive performance of previously constructed cefazolin pharmacokinetic models and determine whether cefazolin administration via the target‐controlled infusion (TCI) method may be possible in clinical practice.

Methods

Twenty‐five gastrectomy patients receiving cefazolin as a prophylactic antibiotic were enrolled. Two grams of cefazolin was dissolved in 50 mL of normal saline to give a concentration of 40 mg mL−1. Before skin incision, cefazolin was administered using a TCI syringe pump, and its administration continued until the end of surgery. The target total plasma concentration was set to 100 μg mL−1. Total and unbound plasma concentrations of cefazolin were measured in three arterial blood samples collected at 30, 60 and 120 min after the start of cefazolin administration. The predictive performance of the TCI system was evaluated using four measures: inaccuracy, divergence, bias and wobble.

Results

Total (n = 75) and unbound (n = 75) plasma concentration measurements from 25 patients were included in the analysis. The pooled median (95% confidence interval) biases and inaccuracies were 6.3 (4.0‐8.5) and 10.5 (8.6‐12.4) for the total concentration model and −10.3 (−16.8 to −3.7) and 22.4 (18.2‐26.7) for the unbound concentration model, respectively. All unbound concentrations were above 10 μg mL−1.

Conclusion

Administration of cefazolin by the TCI method showed a clinically acceptable performance. Applying the TCI method by setting the total concentration as the target concentration rather than the unbound concentration is effective in maintaining a constant target concentration of cefazolin.
A novel link between melatonin and circ_0005753/PTBP1/TXNIP regulatory network in the modulation of osteogenic potential in mesenchymal stem cells
Authors: Tu Wei , Zhu Shuying , Li Fan , Xu Chengyun , Tu Weiping , Chen Yanxia
Published: October 2023

Labeled with pluripotent potential, the transplantation of bone marrow mesenchymal stem cells (BMSCs) is considered as a promising strategy for treating osteoporosis (OP). Melatonin (MEL) has been investigated to be an essential regulator involved in bone metabolism, as well as BMSCs differentiation. Circular RNAs (circRNAs) are a unique kind of non‐coding RNA and play an important regulatory role in OP. However, whether circRNAs are implicated in the effects of MEL on BMSCs osteogenic differentiation remains largely indeterminate. Expression of circ_0005753 in human BMSCs with MEL treatment, clinical specimens diagnosed with OP, either with ovariectomy (OVX)‐induced mice, was measured by RT‐qPCR. Western blot was conducted to analyze protein levels of osteogenesis‐related molecules (Opg, RUNX2, ALP, BMP4) and TXNIP. RNA immunoprecipitation (RIP) and RNA pull‐down assays were performed to validate the binding relationship among circ_0005753, PTBP1, and TXNIP. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were performed to evaluate osteogenic capacity of BMSCs. OP mouse model was established by ovariectomy, as evaluated pathologic changes via hematoxylin–eosin (HE), Masson, and Immunohistochemistry (IHC) staining. Expression of circ_0005753 was remarkably decreased during MEL‐induced osteogenic differentiation of BMSCs. Interestingly, not only circ_0005753 knockdown significantly promoted osteogenic differentiation of BMSCs, but circ_0005753 overexpression also weakened osteogenic differentiation induced by MEL treatment. Mechanistically, circ_0005753 maintained the stabilization of TXNIP mRNA via recruiting PTBP1. Additionally, reinforced circ_0005753 abrogated MEL‐mediated protective effects on OP pathogenesis in a mouse model. This work shows that MEL facilitates osteogenic differentiation of BMSCs via the circ_0005753/PTBP1/TXNIP axis, which may shed light on the development of a novel therapeutic strategy to prevent OP.
Does parent report of child physical abuse and supervisory neglect differ by method of study recruitment or mode of survey administration?
Authors: Freisthler Bridget , Kranich Christiana , Price Wolf Jennifer
Published: October 2023

General population studies find that rates of child maltreatment are higher than those identified in the child welfare system. Some modes of administration may result in higher disclosure rates of child maltreatment by respondents. This study assesses differences in characteristics of parents who are recruited using telephone sampling techniques or via the internet and whether the survey was administered by telephone or the web. We have 1599 parent respondents recruited from listed samples and Craigslist advertisements. Surveys were administered via the telephone or online. Survey included measures of parenting behaviours, psychosocial characteristics and demographics for the parent, child and family. Data were analysed using logit multilevel models. We found no differences between mode of administration and our child maltreatment and parenting measures, including physical abuse, corporal punishment and supervisory neglect. Recruitment strategy was not related to leaving a child home alone and corporal punishment. However, we did find that parents recruited by Craigslist use physical abuse more frequently. That we found no difference in use of punitive and neglectful parenting practices by survey mode of administration and recruitment method provides some degree of confidence that survey methods other than random‐digit‐dialling telephone surveys may result in generalizable samples in this age of declining landline use.
Feasibility of providing web‐based education to patients prior to kidney transplant evaluation
Authors: Kayler Liise K. , Nie Jing , Solbu Anne , Handmacher Matthew , Feeley Thomas H. , Noyes Katia
Published: October 2023

Background

We previously developed web‐based education to be used by patients prior to kidney transplant (KTX) evaluation. The current feasibility study evaluated patients’ intervention uptake and barriers, and staff experiences of the clinic‐wide implementation in preparation for a definitive comparative effectiveness trial.

Methods

Web links and login instructions to view 17 educational videos designed to promote KTX access were delivered via email or text to adults referred to a single transplant center between 10/2020 and 3/2021. Patient barriers were recorded. Non‐completers were allowed to view the resources in the clinic. N = 7 clinic staff were interviewed about their experiences of in‐clinic delivery of the web‐education. Interviews were recorded with field notes and coded using simple content analysis. Patient characteristics and 30‐month KTX access were examined with Chi‐square, t‐tests, and log‐rank tests.

Results

Of 210 patients, 71% completed the self‐education remotely (completers), 16% attempted but did not complete remotely (attempters), and 13% declined the web link invitation (decliners). Implementation barriers included technology access and use difficulties, unstable internet connectivity, limited staff time in clinic to facilitate technology use by patients, and limited technology attentiveness by patients in clinic. In 3‐group comparisons, remote decliners were older with worse estimated posttransplant survival scores, and attempters were younger, more often Medicaid insured, and lived in higher area deprivation; both were more often deemed ineligible for KTX than completers. Between‐group time‐to‐transplantation was non‐significant (p = .571).

Conclusion

The majority of patients accessed the web‐education remotely; however, more vulnerable demographic populations reported greater problems accessing web‐education. In‐clinic delivery was burdensome to staff and patients. Future adaptive implementation strategies are needed to allow for adequate patient education.
Passiflora fieldiana Tillett ex Vanderplank, a new species in subgenus Passiflora supersection Laurifolia from Venezuela: Passifloraceae
Authors: Vanderplank John , Adair Andrew
Published: October 2023

Passiflora fieldiana Tillett ex Vanderplank, a most spectacular passion flower, is fully described for the first time. Its history, distribution and cultivation are discussed.
The role of generative design and additive manufacturing capabilities in developing human–AI symbiosis: Evidence from multiple case studies
Authors: Bendoly Elliot , Chandrasekaran Aravind , Lima Mateus do Rego Ferreira , Handfield Robert , Khajavi Siavash H. , Roscoe Samuel
Published: October 2023

The benefits of additive manufacturing (AM) extend beyond the attributes of physical products and production processes they enable. Experience with AM can augment the way design is approached and can increase opportunities to pivot toward less familiar design tasks. We begin this qualitative study with a natural experiment made possible by an exogenous shock: the COVID‐19 pandemic. Through a three‐stage case study approach using a grounded theory‐building method, we contrast AM usage among a set of firms, half of which pivoted their resources away from their traditional production and toward a response to this shock. We engage in an abductive reasoning approach to consider common threads in AM capabilities that facilitated this pivoting. Our analyses suggest that the advanced use of generative design (GD), a category of computational technologies enabling novel and optimized design, is a critical attribute of these firms that ended up pivoting to make COVID‐related products. Specifically, firms with experience applying this capability demonstrated a unique ability to pivot during this shock and emphasized their valuation of AM‐enabled agility. We revisited these firms 2 years after initial contact and found that GD was associated with higher levels of innovation and was largely viewed by designers as a mechanism driving double‐loop learning. Overall, our study provides insights into the symbiosis between human and artificially intelligent GD, and the role of such symbiosis in advancing AM capabilities.
How mobile is the transnational business elite? Evidence from Swiss banking executives
Authors: Araujo Pedro , Davoine Eric
Published: October 2023

Over the last two decades, a growing literature has examined the emergence of a transnational business elite. However, the pathways of transnational mobility have not been fully characterized. In this article, we use a combination of sequence analysis and the concept of a career script to investigate the geographical mapping and organizational contexts in which transnational mobility occurs. To achieve this, we rely on a database of 186 executives from the 28 largest Swiss banks, as well as 20 interviews with chief human resource officers and 15 interviews with banking executives. Our findings contribute to relativize and differentiate the phenomenon of transnationalization of business elites by underlining the importance of the career context and by identifying distinct interpretations of international career resources according to different types of banks.
Effects of entecavir and tenofovir alafenamide fumarate treatment on renal function in Japanese elderly patients with chronic hepatitis B
Authors: Tanaka Misako , Akahane Takemi , Kawaratani Hideto , Yorioka Nobuyuki , Koizumi Aritoshi , Asada Shohei , Matsuda Takuya , Iwai Satoshi , Tsuji Yuki , Fujinaga Yukihisa , Nishimura Norihisa , Kitagawa Koh , Kaji Kosuke , Namisaki Tadashi , Yoshiji Hitoshi
Published: October 2023

Aim

Entecavir (ETV) and tenofovir alafenamide fumarate (TAF) are considered safe nucleoside/nucleotide analogs (NA) for the kidney. This study aimed to investigate the long‐term effects of ETV or TAF on renal function in elderly patients with chronic hepatitis B (CHB) in Japan.

Methods

The study included 246 CHB patients treated with ETV (184 patients) or TAF (62 patients) for at least 2 years. These patients were divided into two groups: those <65 years of age (130 patients) and those ≥65 years of age (116 patients). The effects of the NAs on renal functions were examined by comparing the estimated glomerular filtration rates (eGFR) from baseline to 2 years between the two groups.

Results

The change in eGFR from baseline to 1 or 2 years after treatment was significantly decreased in both groups. However, the amount of change at 1 and 2 years was significantly greater in the group aged ≥65 years than in the group aged <65 years. The amount of change in eGFR from baseline to 1 and 2 years after treatment was significantly greater in the group aged ≥65 years than in the group aged <65 years, regardless of the type of NA, the prior treatment history, cirrhosis/chronic hepatitis, hypertension, dyslipidemia, and diabetes. Additionally, logistic regression analysis showed that age ≥65 years was independently associated with a decreased eGFR after 2 years of NA treatment.

Conclusions

Long‐term administration of NA to CHB patients over 65 years of age should be carefully monitored for renal impairment.
Fabrication of healthier monoglyceride‐based oleogel containing linseed‐sunflower oil and its application as shortening in cake formulation
Authors: Roufegarinejad Leila , Ahmadi Golnar , İcyer Necattin Cihat , Saıd Toker Omer , Habibzadeh Khiabani Arezou
Published: October 2023

The aim of this work was to fabricate monoglyceride oleogels with combination of linseed (LO) and sunflower oil (SO) and evaluate their effect in baked cakes as a shortening replacer. Physicochemical properties of the oleogels were evaluated from FTIR, Differential scanning calorimetry (DSC), X‐ray diffraction (XRD), morphologic, oil binding, oxidative stability, and fatty acid point of view. Results showed that as the content of linseed oil increases, the ratio of omega‐6 to omega‐3 fatty acids in oleogels decreases. Adding 50% of each oil in the oleogel formulation improved crystallinity, morphological, and intermolecular hydrogen bonding. The 50% SO‐50% LO sample exhibited the highest oil‐binding capacity (89.83 ± 3.51) and peroxide value (1.46 ± 0.26) and was utilised in cake formulations as an optimised sample. Fatty acid, specific volume, porosity, peroxide value, colour, and sensory properties of cakes oleogel were also analysed. The formulated cake with oleogels showed an acceptable peroxide and fatty acid composition. As a result, using oil combinations in oleogel and cake formulations could pave the way for developing foods with a balance ω6/ω3 ratio and reduced levels of saturated and trans fatty acids.
The Effect of Glycolic Acid on Cultured Human Skin Fibroblasts: Cell Proliferative Effect and Increased Collagen Synthesis
Authors: Kim Seong‐Jin , Won Young‐Ho
Published: October 2023

Glycolic acid peeling is known to improve photoaging processes such as wrinkling and roughness, but this effect has not been clearly defined, even though functional activation of fibroblasts has been suggested. The study was aimed to determine the effects of glycolic acid and malic acid (AHA: alpha hydroxy acid) on cultured dermal fibroblasts. Whether it directly increases cell proliferation may be an important factor influencing the production of extracellular matrix such as type I collagen.

Cultured human skin fibroblasts were treated for 24 hours with glycolic acid and malic acid at different concentrations (10−4, 10−5, 10−6 M), and cell proliferation was measured by MTT assay. Then quantitative analysis of collagen synthesis was performed by PICP (Procollagen Type I C‐peptide) enzyme immunoassay and radioisotope (3H‐proline) labelled collagen assay.

The results showed increased cell proliferation and collagen production in response to glycolic acid in a dose dependent manner. The range of cell proliferation and collagen production were significantly higher with glycolic acid treatment than with malic acid or control. It was suggested that the favorable effects of glycolic acid treatment on aging skin were mediated by increased cell proliferation in addition to functional activation of fibroblasts.
Pemphigus Vulgaris Associated with Transient Acanthosis Nigricans Like Lesion
Authors: Usui Keitaro , Kondo Aki , Nakagawa Hidemi
Published: October 2023

A patient with pemphigus vulgaris was treated with predonisolone and cyclosporin. At the sites of healing erosions, hyperkeratotic, hyperpigmented plaques developed; they were clinically and histologically similar to acanthosis nigricans. These lesions regressed after a few months. This case and others described in the literature suggest that such dermatoses might be caused by immunologically as well as mechanically mediated epidermal injury.
Changes in Immunological Parameters after Combination Adjuvant Therapy with Intravenous DTIC, ACNU, and VCR, and Local Injection of IFN‐β (DAV + IFN‐β Therapy) into Malignant Melanoma
Authors: Umeda Tadashi , Aoki Kazuhiro , Yokoyama Akiko , Ohara Hiroyuki , Hayashi Ohmi , Tanaka Kazuyuki , Nishioka Kiyoshi
Published: October 2023

Combination adjuvant therapy with intravenous dimethyl triazeno imidazole carboxamide (DTIC), 1‐[4‐amino‐2‐methyl‐5‐pyrimidinyl]‐methyl‐3‐[2‐chloroethyl]‐3‐nitrosourea hydrochloride (ACNU) and vincristine (VCR) and local injection of interferon‐β (IFN‐β) (DAV + IFN‐β therapy) has been widely applied to treat malignant melanoma, and its therapeutic effect is accepted in Japan. Natural killer (NK) activity, CD4+ and CD8+ T cell counts, CD4/CD8 ratio, and white blood cell counts were analyzed before and after DAV + IFN‐β therapy in order to validate its efficacy. After DAV + IFN‐β therapy, the CD4/CD8 ratio was elevated; however, numbers of both CD4+ and CD8+ T cells and NK activity were consecutively depressed. Peripheral lymphocytes were also decreased, possibly by myelosuppression due to the DAV therapy. The posttreatment suppression of NK activity appeared in spite of the administration of IFN‐β. It is suggested that a more effective adjuvant immunomodulator should be introduced to improve the therapeutic effect of the combination adjuvant chemotherapy in malignant melanoma.
A Case of Nodular Cystic Fat Necrosis: The End Stage Lesion Showing Calcification and Lipomembranous Changes
Authors: Oh Chee‐Won , Kim Kab‐Hyeong
Published: October 2023

Nodular cystic fat necrosis, first described by Przyjemski et al. in 1978, is a distinct, benign subcutaneous lesion characterized histologically by encapsulated fat necrosis. We report a case of nodular cystic fat necrosis in a 22‐year‐old man who had had two mobile, rice‐sized, deep‐seated papules on his right shin for ten years after trauma. Histologically, the excised mass showed encapsulated fat necrosis, calcification, and lipomembranous changes. The encapsulation of necrotic tissue may prevent further extension of adiponecrosis. The subcutaneous fat is prone to trauma or ischemia. The observation of lipomembranous changes in nodular cystic fat necrosis seems to support the concept that lipomembranous change is a nonspecific pattern of the fat necrosis due to multiple local or systemic events causing a compromise in the blood supply of the subcutaneous tissue.
Immobilization of enzymes on poly(vinyl alcohol)‐coated perfluorocarbon supports: comparison of techniques for the immobilization of trypsin and α‐amylase on poly(vinyl alcohol)‐coated solid and liquid perfluorocarbons
Authors: Chase Howard A. , Yang Yuhui
Published: October 2023

An investigation of different methods for the immobilization of α‐amylase and trypsin on solid and liquid poly(vinyl alcohol) (PVA)‐coated perfluorocarbon supports has been made. It is found that very reactive PVA‐coated perfluorocarbon supports are produced only via the reactions between the hydroxy groups of PVA adsorbed on perfluorocarbons and 2, 2, 2‐trifluethanesulphonyl chloride (tresyl chloride), 2, 4, 6‐trichloro‐1, 3, 5‐triazine (cyanuric chloride) or p‐β‐sulphate‐(ethyl sulphonide)‐aniline (SESA), a preactivating reagent for diazotization reactions. The amounts of immobilized trypsin (specific activity and percentage recovery of enzyme activity toward casein) on solid PVA‐coated perfluorocarbon activated by tresyl chloride, SESA or cyanuric chloride were 58 mg/g (1200 units/g, 5.8%), 25 mg/g (1100 units/g, 12.4%) or 44 mg/g (1128 units/g, 7.2%) respectively. The amounts of immobilized α‐amylase (specific activity and the recovery of enzyme activity towards starch) on the same supports were 32 mg/g (4000 units/g, 7.2%), 20 mg/g (4940 units/g, 14.2%) or 29 mg/g (2725 units/g, 5.4%) respectively. Most other methods, i.e. epoxy activation (with epichlorohydrin or 1, 4‐butanediol diglycidyl ether), periodate oxidation, glutaraldehyde coupling, 1, 1'‐carbonyldi‐imidazole chemistry, 1, 1′‐carbonyldi‐imidazole chemistry in an oriented fashion, isothiocyanate chemistry and carbodiimide activation, all resulted in lower degrees of activation of PVA‐coated perfluorocarbon supports and thus correspondingly lower specific activities of the immobilized enzymes. Only the methods with SESA or cyanuric chloride are suitable for enzyme immobilization on PVA‐coated liquid perfluorocarbon emulsions. The amounts of immobilized enzymes and the specific activities of the enzymes on liquid PVA‐coated perfluorocarbon emulsion were, however, lower than those on solid PVA‐coated perfluorocarbon owing to the lower surface area of this carrier. The method with SESA seems to be the best for all applications. The addition of (NH4)2SO4 to the immobilized reactions significantly enhanced the activity recovery and the specific activity of the α‐amylase. The immobilized enzymes were stable during storage. The study demonstrates that solid PVA‐coated perfluorocarbon supports are promising carriers for enzyme immobilization.
Production and characterization of thermostable phenol oxidases of the ascomycete Thermoascus aurantiacus
Authors: Machuca Angela , Aoyama Hiroshi , Durán Nelson
Published: October 2023

The effect of some culture conditions on extracellular phenol oxidase (PO) production by the thermophilic ascomycete Thermoascus aurantiacus was studied. The effect of different lignocellulosic and an aromatic compound (p‐anisidine) as inducers of PO activity was studied in liquid culture. The most effective substrate, wheat bran, induced between 1 and 2 units/ml PO activity when utilized at a concentration of 1.5% (w/v). The type and size of fungal inoculum influenced the PO production. However, the agitation of cultures did not increase PO production. An initial pH between 6 and 8 resulted in a higher PO production. The crude extracts of T. aurantiacus had activities sufficient to oxidize various typical substrates of POs in the absence of H2O2. Higher activities were attained with 2, 2'‐azinobis‐(3‐ethylthiazoline 6‐sulphonate), 2, 6‐dimethoxyphenol and o‐dianisidine. Typical inhibitors of metal‐containing oxidases were the most potent inhibitors of T. aurantiacus PO. The optimal pH for o‐dianisidine oxidation was 2.8 in 50 mM citrate/phosphate buffer; the optimal oxidation temperature was between 70 and 80°C. All characteristics of the crude extract indicated that the PO is a typical fungal laccase, except for the high optimal temperature.
Immobilization of glucose oxidase on poly‐(L‐lysine)‐modified polycarbonate membrane
Authors: Suye Shin‐ichiro , Kumon Yuji , Ishigaki Akira
Published: October 2023

The paper describes the immobilization of glucose oxidase on a polycarbonate membrane modified by a urethane coupling with poly‐(L‐lysine) and activated with glutaraldehyde. The enzymic properties of the immobilized enzyme were investigated and compared with the those of native glucose oxidase. The thermal stability and pH stability of the immobilized glucose oxidase were greater than those of the native enzyme. The membrane was applied as a glucose sensor.
Expression and equilibrium denaturation of cardiac troponin I: stabilization of a folding intermediate during denaturation by urea
Authors: Morjana Nihmat , Tal Rony
Published: October 2023

Human cardiac troponin I has been expressed at high level in Escherichia coli as a fusion protein by using the expression vector Ptac114. The expressed protein forms primarily intracellular inclusion bodies that are solubilized in the presence of 8 M urea. The purified troponin I is recognized by anti‐(human cardiac troponin I) monoclonal antibodies. Equilibrium denaturation of recombinant human troponin I and bovine troponin I is compared by monitoring changes in the protein's fluorescence and CD characteristics. At pH 7·5 the equilibrium denaturation of both proteins by urea occurs in two distinct steps involving at least three major conformational states: native, intermediate and fully denatured. The biphasic profile in the presence of urea is observed by both fluorescence and CD spectroscopy. In the intermediate state the native tertiary structure is largely disrupted and 40% of the secondary structure is conserved, as suggested by near‐UV and far‐UV CD respectively. Thermal denaturation of troponin I, as followed by fluorescence, shows a loss in the signal that is not reversible after heating to 90 °C. In the presence of a constant amount of urea (not greater than 0·5 M) the thermal denaturation becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by urea. The fluorescence of 1‐anilino‐8‐ naphthalenesulphonate produced on binding troponin I decreases in the presence of increasing concentrations of urea up to 3 M; at higher urea concentrations no further change in the remaining signal is observed. Kinetic studies show at least two phases of renaturation for troponin I previously denatured with 8 M urea, whereas only a single phase is detected for the renaturation process in the presence of 3 M urea. The results suggest the occurrence of a stable folding intermediate, the formation of which might be related to the two‐domain architecture of troponin I.
Surface‐induced changes in the structure and activity of enzymes physically immobilized at solid/liquid interfaces
Authors: Norde Willem , Zoungrana Thierry
Published: October 2023

A proteolytic enzyme, α‐chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water‐soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water‐soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat‐induced inactivation process is not identical with the heat‐induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.
Amplification of flow‐microcalorimetry signal by means of multiple bioaffinity layering of lectin and glycoenzyme
Authors: Gemeiner Peter , Dočolomanský Peter , Vikartovská Alica , Štefuca Vladimír
Published: October 2023

This paper demonstrates a positive influence of a special, stepwise technique of enzyme immobilization based on the biospecific adsorption of the glycoenzyme invertase on immobilized concanavalin A (Con A), subsequent adsorption of the free Con A on the immobilized invertase:Con A support and repeated adsorption of invertase on the support. A 3‐fold repetition of the same procedure designed preliminarily as bioaffinity layering afforded up to a 10‐fold increase in catalytic activity of the immobilized invertase. Reactive hydrogels based on bead cellulose and bead poly(glycidyl methacrylate) were used as immobilization supports for the preparation of these highly active preparations. The enhancement in catalytic activity of immobilized invertase preparations was demonstrated thermometrically, by flow microcalorimetry. Further attractive aspects for utilizing the signal amplification of biosensors with immobilized enzymes are discussed.
α‐Galactosidases of Penicillium simplicissimum: production, purification and characterization of the gene encoding AGLI
Authors: Luonteri Elina , Alatalo Edward , Siika‐aho Matti , Penttilä Merja , Tenkanen Maija
Published: October 2023

Production of extracellular a‐galactosidases by the filamentous fungus Penicillium simplicissimum (previously P. janthinellum) VTT‐D‐78090 was studied on different carbon sources. Steam‐exploded oat husks were chosen as the best carbon source for enzyme production. Three a‐galactosidases (AGL) were purified from the culture filtrate using ion‐exchange chromatography, hydrophobic interaction chromatography and gel filtration. The isoelectric points of AGLI, AGLII and AGLIII were 5.2, 4.4 and 7.0, and the molecular masses as determined by SDS/PAGE were 61, 84 and 61 kDa, respectively. All enzymes were glycosylated. The optimum pH for the activity of AGLI and AGLIII was between 3.0 and 4.5 and that of AGLII was between 4.0 and 5.0. AGLII was more stable and more resistant to product inhibition by galactose than the other two enzymes. AGLI and AGLIII were also inhibited by p‐nitrophenol‐a‐D‐galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agl1, encodes 435 amino acids including the signal sequence. It showed similarity with the other a‐galactosidases belonging to the glycosyl hydrolase family 27. The N‐terminal amino acid sequence of AGLIII was also similar to the sequences of other members of family 27, whereas the N‐terminus of AGLII was completely different from the sequences of other reported hydrolases.
Maximization of cholesterol oxidase production by Rhodococcus equi no. 23 by using response surface methodology
Authors: Lee Ming‐Tsung , Chen Wen‐Chang , Chou Cheng‐Chun
Published: October 2023

Medium optimization for the production of cholesterol oxidase (EC 1.1.3.6) by Rhodococcus equi no. 23 was investigated by using response surface methodology and a central composite design. Results revealed that cholesterol and yeast extract had positive effects and the interaction between any two of three factors had no significant effect on cholesterol oxidase production. The optimized medium was the basal medium with the addition of 2.30 g/l cholesterol, 8.18 g/l yeast extract and 4.10 ml/l Tween 80. The peak cholesterol oxidase production (0.242 unit/ml) after 60–72 h cultivation was approx. 4‐fold that in control medium.
Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology
Authors: Kohen Elli , Gatt Shimon , Schachtschabel Astrid , Schachtschabel D.O. , Kohen Cahide , Agmon Vered , Hirschberg Joseph G. , Monti Marco , Roisen Fred
Published: October 2023

The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell‐membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors. Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy. In the multiprobe–multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations. ‘Smart’ instrumentation does not mean ‘smart’ probes. It turns out that the cell's own endogenous probes, the fluorescent coenzymes, may be akin to ‘smart’ probes, open to study in situof many‐fold interrelated pathways in cell energetics and dynamics. Resolution at the micro‐ and even nano‐compartment levels is not altogether impossible. Thus an innovative search in terms of what may be called ‘intracellular reconnaissance with fluorescent probes and biopharmaceuticals’ necessitates recourse to multiple tentative probings along the pleiotropic mechanisms as far in resolution as one can go. Among the characteristic findings using this approach are: (i) morphometric alterations in the mitochondria and melanosomes of melanoma cells treated with azelaic acid; (ii) deregulation of mitochondrial control and extramitochondrial metabolism in similarly treated cells; (iii) considerable acceleration of NAD(P) transient kinetics in atractylate‐treated L sarcoma cells; (iv) alterations of mitochondria and Golgi in fusion‐deficient myoblasts; (v) tentative recognition of β‐glucosidase deficiency in Gaucher disease cells by the use of fluorescent and fluorogenic lysosomal probes; and (vi) UVA‐induced accumulation of Schiff bases (a kind of accelerated photo‐aging) in yeast and kidney epithelial cells. Because these studies utilize probing at whatever points along the concerned pathways become accessible, at first glance they may look disconnected. What and where is the connecting thread, for instance, between studying melanoma metabolism, melanosome morphometry, hepatocyte organelle morphogenesis and transformation, myotube organelle morphogenesis and fusion–non‐fusion, and lysosomal activity in gene‐deficient cells? In the mapping of the regulatory and deregulatory mechanisms involved in the switching of differentiation or transformation, each of the above topics carries an information content towards resolution of the pleiotropic puzzle. The integration of such information with increasing resolution and access to intracellular microdomains may ultimately allow focus on the precise target, the switch from differentiation to transformation or vice versa.
Cofactor recycling in a coupled enzyme oxidation–reduction reaction: conversion of ω‐oxo‐fatty acids into ω‐hydroxy and dicarboxylic acids
Authors: Nuñez Alberto , Foglia Thomas A. , Piazza George J.
Published: October 2023

Aldehydes are reduced to alcohols by the enzyme alcohol dehydrogenase (ADH), whereas the enzyme aldehyde dehydrogenase (AldDH) oxidizes aldehydes to carboxylic acids. ADH and AldDH require, respectively, the reduced and oxidized forms of the cofactor NAD (NAD+/NADH). By combining both oxidation and reduction reactions into one process, it is possible to produce alcohols and carboxylic acids simultaneously from aldehydes by continuous recycling of the NAD+/NADH cofactor. However, both enzymes need to be active within the same pH region and buffer system. To test this hypothesis, the pH profile (Vmax and Vmax/Km) as well as the pKa of the prototropic groups involved in catalysis for both dehydrogenases were determined using (Z,Z)‐nona‐2,4‐dienal as a model substrate. The pH profile (Vmax and Vmax/Km) of both enzymes overlapped in the pH range of 6–8 in potassium phosphate buffer. When the coupled enzyme system was used at pH 7 with 10% NAD+ cofactor, over 90% of the starting aldehyde was converted to its corresponding acid and alcohol derivatives in a 1:1 ratio. The sequential action of the enzymes lipoxygenase and hydroperoxide lyase converts polyunsaturated fatty acids to aldehydic fatty acids. The products arising from the oxidation or reduction of the aldehydic functionality are of industrial interest. It was found that 13‐oxo‐9‐(Z),11‐(E)‐tridecadienoic acid, the product of the sequential reaction of soya bean lipoxygenase and hydroperoxide lyase from Chlorella pyrenoidosaon linoleic acid, is also a substrate in this coupled enzyme system.
Effect of α‐tocopherol and silibin dihemisuccinate on the proliferation of human skin fibroblasts
Authors: Onat D. , Boscoboinik D. , Azzi A. , Basaga H.
Published: October 2023

Cell proliferation is a complex and important event in atherosclerosis, aging and cancer, and is under the control of signalling pathways. These signalling pathways in turn are effected by the presence of a number of chemicals. For this purpose, we have checked the effect of two chemicals on the proliferation of skin fibroblasts. α‐Tocopherol and silibin dihemisuccinate (SDH) negatively regulate proliferation of human skin fibroblasts. To check the cell‐cycle time intervals, a [3H]thymidine incorporation assay was performed, showing DNA replication at around 24 h; this indicated the time required for the incubation with the chemicals. When α‐tocopherol was added to the growth medium at a physiological concentration of 50 μM, cell proliferation was inhibited by 40% in 72 h. A similar inhibitory effect of cell proliferation was achieved when 500 μM SDH was used (39% inhibition in 72 h). From the dose–response curves obtained it was concluded that both duration of treatment and the concentration of the chemicals are important parameters. The actual mechanism of the inhibition of cell proliferation may be due to the anti‐oxidative potential of these chemicals as well as another mechanism effecting signal transduction pathways.
Good manufacturing practice (GMP) compliance in the biologics sector: plasma fractionation
Authors: Ways Judy P. , Preston M. Sue , Baker Donald , Huxsoll Jean , Bablak Jason
Published: October 2023

The U.S. blood supply is the safest it has ever been. Due to blood safety and the introduction of viral inactivation/clearance technologies, protein therapies derived from human blood have also in recent years had a history of product safety. Nevertheless, since 1995, the plasma‐fractionation industry has experienced increased compliance‐related actions by the Food and Drug Administration (FDA), as shown by a substantive increase in the number of FDA 483 inspectional observations, FDA warning letters and other FDA regulatory action. An evaluation of these trends shows that they reflect the implementation by the FDA of increased inspectional interest in the plasma‐fractionation industry and an evolution of inspectional practices and standards of current good manufacturing practice (cGMP). Plasma fractionators have responded to FDA actions by carefully evaluating and addressing each inspectional observation, assessing impact to product and taking appropriate actions, including corrective actions to prevent future occurrence. They have made major investments in facilities, quality systems, personnel and training to meet the evolving standards of cGMP and in an effort to implement these standards systemically. Through industry associations, manufacturers have further enhanced product safety by adopting additional voluntary standards for plasma to prevent the entry of potentially unsuitable plasma into the production process. The industry remains committed to application of cGMP and to working with the FDA in further evolution of these standards while striving to assure a continued supply of safe, pure and effective plasma‐derived therapies.
Production of carcinoembryonic antigen (CEA) N‐A3 domain in Pichia pastoris by fermentation
Authors: Hellwig Stephan , Robin Frederic , Drossard Jürgen , Raven Nicole P. G. , Vaquero‐Martin Carmen , Shively John E. , Fischer Rainer
Published: October 2023

Carcinoembryonic antigen (CEA) is a 180‐kDa glycoprotein found on the surface of normal colon and malignant human adenocarcinomas. Recently, a fusion protein containing two of the seven Ig‐like domains present in CEA (N and A3) has been constructed and expressed in Pichia pastoris [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193–3201]. Here, we report the generation and selection of a multi‐copy clone expressing this fusion protein, the optimization of the shake‐flask expression protocol and the upscaled production of CEA N‐A3 using fermentation technology. P. pastoris transformants secreting the CEA N‐A3 domain were generated by electrotransformation of the GS115 host strain with the pPIC9K vector containing the CEA N‐A3 cDNA [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193–3201] then screened for CEA N‐A3 expression and G418 resistance. The recombinant CEA N‐A3 domain was detected in the culture supernatant using the monoclonal anti‐CEA antibody T84.66. Optimization of methanol‐induction conditions resulted in a high‐methanol shake‐flask expression protocol yielding significantly increased CEA N‐A3 levels. Fermentation and culture conditions were optimized for 5‐l working‐volume fermentations and CEA N‐A3 was affinity purified using Ni‐IDA (imino di‐acetic acid) affinity chromatography from the clarified fermentation supernatant. Peptide N‐glycosidase F treatment revealed that the recombinant protein was heavily glycosylated but expressed as a single polypeptide of 28 kDa with no evidence of proteolytic degradation. Our results demonstrate that functional CEA N‐A3 domain can be produced in sufficient quantities in P. pastoris for structural analysis or diagnostic applications. To our knowledge, this article represents the first report on the production of a human tumour antigen through fermentation.
Towards molecular farming in the future: transient protein expression in plants
Authors: Fischer Rainer , Vaquero‐Martin Carmen , Sack Markus , Drossard Jürgen , Emans Neil , Commandeur Ulrich
Published: October 2023

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.
Towards molecular farming in the future: Pichia pastoris‐based production of single‐chain antibody fragments
Authors: Fischer Rainer , Drossard Jürgen , Emans Neil , Commandeur Ulrich , Hellwig Stephan
Published: October 2023

This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein‐expression system. P. pastoris is a useful system for the expression of milligram‐to‐gram quantities of a protein, which can be scaled up to fermentation to meet greater demands. Compared with mammalian cells, Pichia do not require a complex growth medium or culture conditions, they are as easy to manipulate genetically as Escherichia coli and have a eukaryotic protein‐synthesis pathway. They seem suited to laboratory‐scale production of recombinant proteins for in‐house use or, in some cases, molecular farming of recombinant products. This review article focuses on the use of P. pastoris, describes a fermentation production run of a single‐chain antibody fragment and includes a discussion of fermentation as a production strategy.
Women in Cultural Transition: Suicidal Behavior in South African Indian Women
Authors: Wassenaar Douglas R. , van der Veen Marchiene B. W. , Pillay Anthony L.
Published: October 2023

Young South African Indian women are a population with relatively high rates of suicidal behavior. This article presents three illustrative case studies of nonfatal suicidal behavior by South African Indian women. The cases are discussed in relation to a review of epidemiological, historical, and sociocultural perspectives on South African Indian women. It is argued that suicidal behaviors in this group can be understood in the context of sociocultural transition. Transitional tensions between traditional Indian culture and Westernization have an impact on traditional gendered power relations and generate conflicts that have intrapsychic and interpersonal consequences for women and for men. These in turn may have adverse effects on marital functioning, quality of life, and specific aspects of emotional functioning, which have been identified as precursors of suicidal behavior. The case studies are discussed with reference to gender narratives. Implications for prevention, intervention, and future research are discussed.
Conceptions of Death and Suicide in Children Ages 6–12 and Their Implications for Suicide Prevention
Authors: Mishara Brian L.
Published: October 2023

Interviews were conducted with 65 public school children in Grades 1–5 concerning their understanding of and experiences with death and suicide, and investigating the development of the Piagetian concepts of life and age. By third grade, children have an elaborate understanding of suicide, and younger children generally understand “killing oneself,” although their understanding of death and living may be immature. Children learn about suicide from television and discussions with other children, but they rarely discuss suicide with adults. The level of development of the concept of suicide is related to maturity rather than specific experiences. Implications for primary prevention are discussed.