bluuebunny/crossref_metadata_2025 huggingface dataset:
Intermittently scanned continuous glucose monitoring in adults with type 1 diabetes: A subgroup analysis from the FLASH‐UK study
Authors: Leelarathna Lalantha , Sutton Christopher J. , Evans Mark L. , Neupane Sankalpa , Rayman Gerry , Lumley Sarah , Cranston Iain , Narendran Parth , Krishan Ashma , Taxiarchi Vicky P. , Barnard‐Kelly Katharine , Elliott Rachel A. , Burns Matthew , Camm Maisie , Thabit Hood , Wilmot Emma G. ,   the FLASH‐UK Trial Study Group
Published: October 2023

Aims

The FLASH‐UK trial showed lower HbA1c with intermittently scanned continuous glucose monitoring (isCGM), as compared with self monitoring of blood glucose (SMBG), in adults with type 1 diabetes and HbA1c ≥58 mmol/mol (≥7.5%). Here, we present results from the pre‐specified subgroup analysis for the 24‐week HbA1c (primary outcome) and selected sensor‐based secondary outcomes.

Methods

This was a multi‐centre, parallel‐design, randomised controlled trial. The difference in treatment effect between subgroups (baseline HbA1c [≤75 vs. >75 mmol/mol] [≤9.0 vs >9.0%], treatment modality [pump vs injections], prior participation in structured education, age, educational level, impaired awareness of hypoglycaemia, deprivation index quintile sex, ethnic group and Patient Health Questionnaire‐9 [PHQ‐9] detected depression category) were evaluated.

Results

One hundred fifty‐six participants (females 44%, mean [SD] baseline HbA1c 71 [9] mmol/mol 8.6 [0.8%], age 44 [15]) were randomly assigned, in a 1:1 ratio to isCGM (n = 78) or SMBG (n = 78). The mean (SD) baseline HbA1c (%) was 8.7 (0.9) in the isCGM group and 8.5 (0.8) in the SMBG group, lowering to 7.9 (0.8) versus 8.3 (0.9), respectively, at 24 weeks (adjusted mean difference −0.5, 95% confidence interval [CI] −0.7 to −0.3; p < 0.001]. For HbA1c, there was no impact of treatment modality, prior participation in structured education, deprivation index quintile, sex or baseline depression category. The between‐group difference in HbA1c was larger for younger people (a reduction of 2.7 [95% CI 0.3–5.0; p = 0.028] mmol/mol for every additional 15 years of age). Those with HbA1c 76–97 mmol/mol (>9.0%–11.0%) had a marginally non‐significant higher reduction in HbA1c of 8.4 mmol/mol (3.3–13.5) compared to 3.1 (0.3–6.0) in those with HbA1c 58–75 mmol/mol (p = 0.08). For ‘Time in range’ (% 3.9–10 mmol/L), the difference was larger for those with at least a bachelor's degree. For ‘Time below range’ (% <3.9 mmol/L), the difference was larger for those using injections, older people and those with less than bachelor's degree.

Conclusions

Intermittently scanned continuous glucose monitoring is generally effective across a range of baseline characteristics.
Clinical and immunological characteristics of children diagnosed with—Type 1 diabetes during the COVID‐19 pandemic
Authors: Margolis Merav Gil , Weizman Sarit , Lazar Liora , Yakobovich‐Gavan Michal , Tenenbaum Ariel , Phillip Moshe , Oron Tal
Published: October 2023

Aims

To find clinical and immunological signatures of the SARS‐CoV‐2 and the COVID‐19 pandemic on children newly diagnosed with type 1 diabetes (T1D).

Methods

A single‐centre, retrospective, observational study comparing the clinical and immunological characteristics of children diagnosed with T1D the year before and during the first 2 years of the COVID‐19 pandemic. Data extracted from the medical records included clinical and demographic parameters, COVID‐19 PCR results and the presence of anti‐islet, thyroid and celiac‐related antibodies. Also obtained from the medical records was a family history of T1D, celiac disease and autoimmune thyroid disease in a first‐degree family member.

Results

A total of 376 children were diagnosed with T1D during the study period. A total of 132 in the pre‐COVID era and 246 in the first 2 years of the pandemic. At diagnosis, the pH in children with DKA was lower, and HbA1c tended to be higher in the COVID‐19 group compared to the pre‐COVID‐19 group (7.30 [7.18, 7.35] vs 7.33 [7.19, 7.36], p = 0.046) and (110.9 [86.9, 129.5] vs 100 [80.3, 129.5], p = 0.067]) respectively. Multiple islet antibodies (IA) were significantly more common among patients in the pre‐COVID‐19 group compared to the COVID‐19 group (72% vs 61%, p = 0.032). Tissue transglutaminase antibodies were more common among children diagnosed in the COVID‐19 compared to the pre‐COVID group (16.6% vs 7.9%, p = 0.022).

Conclusions

Our findings suggest that SARS‐CoV‐2 and the environmental alterations caused by the pandemic affected the clinical characteristics and the immunological profile of children diagnosed with T1D. It is, therefore, plausible that the virus plays a role in the autoimmune process causing T1D.
The lasting impact of external shocks on political opinions and populist voting
Authors: Levi Eugenio , Sin Isabelle , Stillman Steven
Published: October 2023

We use electoral survey data linked to disaggregated geographical data to examine the impact that two external shocks had on the initial development and long‐term success of New Zealand First (NZF), one of the oldest populist parties in the Organisation for Economic Co‐operation and Development, as well as their short and long‐run impact on voting and political opinions. We find that people exposed to both structural and immigration reforms were more likely to initially vote for NZF and permanently changed their political attitudes and policy preferences. Exposure to these shocks plays an important role in explaining the rise and continued success of populism in New Zealand.
‘Speed‐ageing’ of human skin in serum‐free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5‐dimethylpyrazine
Authors: van Lessen Max , Mardaryev Andrei , Broadley David , Bertolini Marta , Edelkamp Janin , Kückelhaus Maximilian , Funk Wolfgang , Bíró Tamás , Paus Ralf
Published: October 2023

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ‐cultured in a defined serum‐free medium. Quantitative (immuno‐)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing‐associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin‐1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA‐damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT‐PCR of whole skin RNA extracts also showed up‐regulated mRNA levels of several (inflamm‐) ageing biomarkers (MMP‐1, ‐2, ‐3, ‐9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti‐ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up‐regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti‐ageing effects of topical 2,5‐dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant “speed‐ageing” assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well‐suited to identify novel anti‐ageing actives directly in the human target organ.
Parenting in the “extreme”: An exploration into the psychological well‐being of long‐term adoptive mothers
Authors: Kohn Charmaine , Pike Alison , de Visser Richard O.
Published: October 2023

Objective

The objective of this study was to increase understanding of factors that influence adoptive mothers' psychological and emotional well‐being. The aims were to compare mothers' self‐reported emotional state 12 months after child placement to the time of the interview and to identify factors that challenge and support maternal mental health.

Background

Mothers who adopt children face unique challenges that put them at a heightened risk of negative mental health. There is a need for better understanding of the influences on their psychological and emotional well‐being.

Method

This qualitative study involved nine in‐depth semistructured interviews. Transcripts were analyzed using reflexive thematic analysis.

Results

The themes offered broad insights, including the consistency of negative mental health across time associated with demands of “extreme parenting,” strains within and across relationships, and deprioritizing self‐care. Factors associated with positive mental health included development of love; attachment and sense of family identity; learning, competence, and “therapeutic parenting,” and the ability to “offload.”

Conclusion

The emotional and psychological well‐being of mothers who adopt children should be validated and recognized as important to the overall adjustment of adoptive families.

Implications

Adoption agencies should take a proactive approach to addressing the mental health needs of adoptive mothers, providing comprehensive information, ongoing support, and regular assessments.
Fish know no borders—Implications of the genetic structure and mixed‐stock composition to cross‐border management of adfluvial brown trout
Authors: Huusko Ari , Nikula Raisa , Tanhuanpää Pirjo , Koljonen Marja‐Liisa , Leinonen Tuomas
Published: October 2023

Effective management of migrating fish stocks requires an understanding of population structure and each stock's contribution to the fishery. Water bodies spanning across national borders pose a particular challenge. We investigated the genetic structure of 13 adfluvial brown trout populations from natal rivers and conducted a genetic mixed‐stock analysis of brown trout from the cross‐border Lake Pyaozero Basin in eastern Fennoscandia, using 16 microsatellite loci. Our analysis revealed a clear genetic structure of three distinct geographic groups, consisting of river‐specific populations. The Western and Southern groups, which included 11 rivers, contributed over 90% to the mixed stock in the lacustrine rearing area. This mixed stock of several river‐specific subpopulations renders poorly managed fishing in the area a significant threat to the entire native adfluvial brown trout population complex.
Does non‐native diversity mirror Earth's biodiversity?
Authors: Briski Elizabeta , Kotronaki Syrmalenia G. , Cuthbert Ross N. , Bortolus Alejandro , Campbell Marnie L. , Dick Jaimie T. A. , Fofonoff Paul , Galil Bella S. , Hewitt Chad L. , Lockwood Julie L. , MacIsaac Hugh J. , Ricciardi Anthony , Ruiz Gregory , Schwindt Evangelina , Sommer Ulrich , Zhan Aibin , Carlton James T.
Published: October 2023

Aim

Human activities have introduced numerous non‐native species (NNS) worldwide. Understanding and predicting large‐scale NNS establishment patterns remain fundamental scientific challenges. Here, we evaluate if NNS composition represents a proportional subset of the total species pool available to invade (i.e. total global biodiversity), or, conversely, certain taxa are disproportionately pre‐disposed to establish in non‐native areas.

Location

Global.

Time period

Present day.

Major taxa studied

Global diversity.

Methods

We compiled one of the most comprehensive global databases of NNS (36,822 established species) to determine if NNS diversity is a representative proportional subset of global biodiversity.

Results

Our study revealed that, while NNS diversity mirrors global biodiversity to a certain extent, due to significant deviance from the null model it is not always a representative proportional subset of global biodiversity. The strength of global biodiversity as a predictor depended on the taxonomic scale, with successive lower taxonomic levels less predictive than the one above it. Consequently, on average, 58%, 42% and 28% of variability in NNS numbers were explained by global biodiversity for phylum, class and family respectively. Moreover, global biodiversity was a similarly strong explanatory variable for NNS diversity among regions, but not habitats (i.e. terrestrial, freshwater and marine), where it better predicted NNS diversity for terrestrial than for freshwater and marine habitats. Freshwater and marine habitats were also greatly understudied relative to invasions in the terrestrial habitats. Over‐represented NNS relative to global biodiversity tended to be those intentionally introduced and/or ‘hitchhikers’ associated with deliberate introductions. Finally, randomness is likely an important factor in the establishment success of NNS.

Main conclusions

Besides global biodiversity, other important explanatory variables for large‐scale patterns of NNS diversity likely include propagule and colonization pressures, environmental similarity between native and non‐native regions, biased selection of intentionally introduced species and disparate research efforts of habitats and taxa.
A secret thing: Forgetting the author in Annette von Droste‐Hülshoff's “Das erste Gedicht”
Authors: Gutterman Julia
Published: October 2023

This article examines the notion of secrecy in Annette von Droste‐Hülshoff's poem “Das erste Gedicht” (1846), pursuing its inquiry through a critical analysis of the gendering of space and authorship in Walter Benjamin's commentary on her poetics inDeutsche Menschen. Eine Folge von Briefen (1936). Critics have understood “Das erste Gedicht” as an autobiographical and exemplary expression of Droste‐Hülshoff's authorial origins in secrecy and shame. By contrast, my article shows how the poem foregrounds forgetting rather than remembering as a poetological figure, thus challenging the notion that authorship originates in a singular past. In dismantling conventional ideas about the relation among time, memory, and writing, Droste‐Hülshoff seems to anticipate literary modernism. Drawing on her manuscripts and letters, this article further shows how the poem's literary genesis reveals a critical engagement with shame and secrecy as gendered affects conventionally attributed to women writers in mid‐19th‐century Germany.
A systematic review of Radiesse/calcium hydroxylapatite and carboxymethylcellulose: evidence and recommendations for treatment of the face
Authors: Guida Stefania , Galadari Hassan
Published: October 2023

Radiesse® is a filler composed of calcium hydroxylapatite microspheres suspended in a carboxymethylcellulose gel (CaHA/CMC). It has robust rheological properties that have been associated with its versatility. CaHA/CMC is employed for both on‐label indications of filling wrinkles or lines, volumizing, and contouring of areas as well as off‐label indications aiming at biostimulation and skin tightening. However, despite the expanding use of CaHA/CMC, overall evidence and recommendations for treatment are currently lacking. This paper aims to provide an up‐to‐date overview of CaHA/CMC clinical applications, together with a level of evidence of supporting literature, focusing on the face. Based on the data, CaHA/CMC may be considered a safe and effective treatment option for cheeks, jawline, HIV‐related facial lipoatrophy, and nasolabial folds. Treatment of marionette lines, chin, pre‐jowl, and corner of the mouth also tends to respond with a high degree of efficacy. Despite the recent trend, guidelines, and safety profile of diluted and hyperdiluted Radiesse®, no randomized controlled trials have been published.
The Use of Apligraf in Acute Wounds
Authors: Kirsner Robert S.
Published: October 2023

Advances in the development of tissue engineered skin have led to several new products aimed at improving wound healing. Designed as a skin substitute, both epidermal and dermal equivalents have been under investigation and used clinically for over a decade. A novel bi‐layered living skin equivalent, Apligraf, has been approved in several countries and is approval in the US FDA for the treatment of venous ulcers. Derived from foreskin, cultured neonatal fibroblasts are combined with bovine type I collagen to form a neodermis over which cultured neonatal keratinocytes are placed, proliferate and differentiate. In addition to use in venous ulcers, Apligraf use has also been reported in acute wounds such as surgical excision sites and partial thickness donor sites. Apligraf's use in acute wounds has lent insight into its biologic behavior, safety and efficacy. Through these experiences, it has been learned that Apligraf behaves similar to a partial thickness autograft, and is safe and effective. In addition, Apligraf appears to ‘take‘, in many cases persists, is not clinically rejected but its ultimate fate is not known.
Seasonal variations in the quality of mopane leaves as a source of browse for mammalian herbivores
Authors: STYLES C. V. , SKINNER J. D.
Published: October 2023

This paper deals with seasonal variations in the chemical composition of mopane leaves in terms of their palatability to herbivores. Green mopane leaves were found to be most palatable over summer, a period when, due to very poor rainfall, the veld was at its most desolate. Senescing leaves were at their most palatable in late winter/early spring, this resource maintaining many herbivores for the short period before the spring leaf flush. Four leaf classes were identified during the spring leaf flush: young green leaves were the most palatable followed by very young leaves, mature green leaves and senescent leaves. The flush is independent of rain and coincides with the end of the dry season, a period when most tree species occurring in association with the mopane are leafless and the grass sward is very poor. It begins earlier on heavily browsed trees. Most ungulates present in the area were found to utilize the nutritious young leaves. The importance of this leaf flush is discussed with respect to the management of the area, and the importance of the heavy pressure large mammals are imposing on this veld type is elicited.
Viruses in Salix grown for bioenergy
Authors: Kopp R. F. , Castello J. D. , Abrahamson L. P.
Published: October 2023

A study was completed to determine if willows propagated vegetatively for bioenergy in New York State were infected by viruses. Unrooted, dormant cuttings from 10 willow clones were planted in a greenhouse. Four months later the young foliage and roots were tested for tomato mosaic tobamovirus, tobacco necrosis necrovirus (TNV), and brome mosaic bromovirus by double‐antibody sandwich enzyme‐linked immunosorbent assay. All three viruses were detected in the roots of some plants, but rarely in foliage. Every clone was infected with at least one virus. Tobacco mosaic virus and TNV were also transmitted to Chenopodium quinoa from extracts of roots collected from field‐grown willows. Imported willow material may be virus‐infected.
Virulence of two Finnish Gremmeniella abietina types (A and B)
Authors: Terho M. , Uotila A.
Published: October 2023

Thirteen Finnish Gremmeniella abietina isolates of types A and B were compared for differences in their virulence. Three kinds of inoculations were made: one with conidia on young Scots pine (Pinus sylvestris) seedlings and two with mycelia on the stems and shoots of young Scots pine trees. Inoculations with conidia were carried out in August 1992 and inoculations with mycelia were carried out six times between 11 August and 20 October, 1992. The experiments were evaluated in the late spring and early summer of 1993. The results showed that there was a difference in virulence between the two types. In the conidial inoculations type A infected 34.7% and type B infected 11.0% of the inoculated seedlings. For mycelial inoculations with type A the mean canker (stems) and necrosis (shoots) lengths were 19.3 mm and 8.6 mm longer, respectively, than with type B inoculations. In shoot inoculations there also was a clear difference between the two types in the number of such inoculations where no symptoms were observed. For type B shoot inoculations there was no fungal growth in 21.5% whereas for type A inoculations the figure was only 3.7%.
Wound response of Eucalyptus clones after inoculation with Cryphonectria cubensis
Authors: van Zyl L. M. , Wingfield M. J.
Published: October 2023

Twenty‐five different Eucalyptus grandis clones were artificially wounded and inoculated with a virulent isolate of Cryphonectria cubensis. The capacity of wounds to close through callus production was correlated with the relative susceptibility of these clones to infection by C. cubensis. Clones with the greatest capacity to close wounds were those that were also most tolerant to C. cubensis infection. Those with a lower capacity to close wounds were most susceptible to Cryphonectria canker.
Mechanistic studies of the oxidation of a non‐phenolic lignin model compound by the laccase/1‐hydroxybenzotriazole redox system
Authors: Li Kaichang , Helm Richard F. , Eriksson Karl‐Erik L.
Published: October 2023

The use of a laccase‐mediator system is one of the promising possibilities for an environmentally benign pulp‐bleaching process. Thus a better understanding of the mechanism for the oxidation of lignin by a laccasemediator system would facilitate the commercialization of such a system for pulp bleaching. By using 1‐hydroxybenzotriazole (1‐HBT) as a mediator, it was found to be partly regenerated during the oxidation of a lignin dimer, 1‐(3, 4‐dimethoxyphenyl)‐2‐(2‐methoxyphenoxy)propan‐1, 3‐diol (I). A free radical of 1‐HBT, generated by laccase, was probably responsible for the oxidation of I. The free radical of 1‐HBT was, however, transformed to benzotriazole, which could not mediate the oxidation of I. The hypothesis that 1‐HBT acted as an activator or a co‐substrate for laccase was not confirmed. Active oxygen species such as hydroxyl free radical (HO•) and superoxide (O2•‐) were also found not to be involved in the oxidation of I.
Chitosan‐membrane interactions and their probable role in chitosan‐mediated transfection
Authors: Venkatesh Savitha , Smith Timothy J.
Published: October 2023

Recent studies in our laboratory have shown that chitosan, a polycationic polymer of glucosamine, can facilitate the transfection of HeLa cells with a plasmid that codes for β‐galactosidase. Although chitosan can bind to DNA and other polyanions, the kinetics of complexation might differ depending on the polyanion tested. This evidence suggests that, in addition to ionic interactions, the carbohydrate backbone of chitosan might have an important role in the process of transfection. Beads prepared by the complexation of chitosan with polyphosphate were used to investigate the nature of cellular interactions with chitosan. HeLa cells bound to chitosan‐polyphosphate beads could be readily displaced from the beads with methyl α‐D‐mannopyranoside but not with NaCl. Membrane proteins solubilized by CHAPS bound readily to chitosan‐polyphosphate beads. A major fraction of the membrane proteins could be eluted from the beads with methyl α‐D‐mannopyranoside. These results suggest that non‐ionic interactions between the carbohydrate backbone of chitosan and cell surface proteins might have an important role in the chitosanmediated transfection of HeLa cells.
Introduction of potential helix‐capping residues into an engineered helical protein
Authors: Parker Matthew H. , Hefford Mary Alice
Published: October 2023

MB‐1 is an engineered protein that was designed to incorporate high percentages of four amino acid residues and to fold into a four‐ α‐helix bundle motif. Mutations were made in the putative loop I and III regions of this protein with the aim of increasing the stability of the helix ends. Four variants, MB‐3, MB‐5, MB‐11 and MB‐ 13, have replacements intended to promote formation of an ‘N‐ capping box’. The loop I and III sequences of MB‐3 (both GDLST) and MB‐11 (GGDST) were designed to cause αL C‐terminal ‘capping’ motifs to form in helices I and III. MB‐5 has a sequence, GPDST, that places proline in a favourable position for forming β‐turns, whereas MB‐13 (GLDST) has the potential to form Schellman C‐capping motifs. Size‐exclusion chromatography suggested that MB‐1, MB‐3, MB‐5, MB‐11 and MB‐13 all form dimers, or possibly trimers. Free energies for the unfolding of each of these variants were determined by urea denaturation, with the loss of secondary structure followed by CD spectroscopy. Assuming an equilibrium between folded dimer and unfolded monomer, MB‐13 had the highest apparent stability (40.5 kJ/mol, with ±2.5 kJ/mol 95% confidence limits), followed by MB‐11 (39.3±5.9 kJ/mol), MB‐ 3 (36.4±1.7 kJ/mol), MB‐5 (34.7±2.1 kJ/mol) and MB‐1 (29.3±1.3 kJ/mol); the same relative stabilities of the variants were found when a folded trimer to unfolded monomer model was used to calculate stabilities. All of the variants were relatively unstable for dimeric proteins, but were significantly more stable than MB‐1. These findings suggest that it might be possible to increase the stability of a protein for which the three‐dimensional structure is unknown by placing amino acid residues in positions that have the potential to form helix‐ and turn‐stabilizing motifs.
Renaturation of recombinant human neurotrophin‐3 from inclusion bodies using a suppressor agent of aggregation
Authors: Suenaga Masato , Ohmae Hiroaki , Tsuji Shinji , Itoh Takashi , Nishimura Osamu
Published: October 2023

Escherichia coli has been widely used in the production of recombinant proteins. One of the drawbacks inherent in this method is that the proteins produced in the cells often form inactive inclusion bodies. Usually, the inclusion bodies can be separated from other cell components, solubilized by denaturants such as guanidine hydrochloride or urea, and then renatured through a refolding process such as dilution or dialysis. However, it has been shown that biologically active recombinant human neurotrophin‐3 cannot be obtained at high yield by this procedure due to aggregation and precipitation of the protein. We applied the refolding process using the aggregation suppressor L‐arginine in the renaturation of neurotrophin‐3, and obtained biologically active neurotrophin‐3 at high yield from the inclusion bodies. Consequently, about 10 mg of purified neurotrophin‐3 was prepared from 1 litre of culture broth.
Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme
Authors: Niles Andrew L. , Maffitt Mark , Haak‐Frendscho Mary , Wheeless Christine J. , Johnson David A.
Published: October 2023

Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin‐like serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two‐step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post‐translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.
Capture of anti‐(Galα1‐3Gal) antibodies by immobilized Galα1‐3Gal oligomers derived from carrageenan
Authors: Klein Elias , Euler Marco , Vercellotti John
Published: October 2023

An immobilized Galα1‐3Gal‐bearing affinity ligand was prepared as a possible prophylactic binding site of human anti‐(Galα1‐3Gal) antibodies for the prevention of hyperacute rejection of pig xenografts. λ‐Carrageenan, a natural polysaccharide known to possess alternately α1‐3 and β1‐4‐linked D‐galactopyranose sulphate residues, was selectively degraded by acetolysis and subsequently deacetylated to obtain λ‐carrageenan oligosaccharides. The resulting syrup was ultrafiltered to remove higher polymers and chromatographed on Sephadex G‐15 to produce a series of sized oligosaccharide fractions. An immuno‐dot‐blot method was established on the basis of the competition between the λ‐carrageenan oligosaccharides in solution and bovine thyroglobulin (BTG), a glycoprotein known to express the Galα1‐3Gal marker, as the solid‐phase antigen. Increasing amounts of λ‐carrageenan fractions added to normal human plasma resulted in a progressive decrease in Galα1‐3Gal‐specific human IgM and IgG binding to BTG. Complete inhibition of Galα1‐3Gal‐specific human immunoglobulin was attained at concentrations less than 0.6 mg of carbohydrate/ml of human plasma with the more active fractions. These bioactive λ‐carrageenan oligosaccharides were immobilized on hydrazide‐modified microporous nylon membranes, yielding capacities between 2.18 and 2.86 mg/ml of membrane. A subsequent decrease in the human anti‐(Galα1‐3Gal) antibody level in normal human plasma was demonstrated and proved with the established immuno‐dot‐blot procedure.
Conversion of cyclodextrin into high‐amylose starch of low molecular mass by means of cyclodextrin glucanotransferase
Authors: Rendleman Jacob A. , Knutson Clarence A.
Published: October 2023

α‐Cyclodextrin (CD) was converted by Bacillus macerans cyclodextrin glucanotransferase into highly insoluble high‐amylose starch with a low average degree of polymerization (56–73), in yields as high as 78% over a wide range of temperatures (25–70 °C). Ability to undergo this conversion was highly concentration‐dependent. γ‐CD was also convertible in good yield; however, β‐CD was relatively resistant to conversion. Degrees of polymerization and the percentages of amylose in the high‐amylose products were estimated from spectrophotometric measurements on their iodine complexes. A plausible mechanism for conversion of CD into starch is proposed.
Purification and characterization of a regiospecific lipase from Aspergillus terreus
Authors: Yadav Raman P. , Saxena Rajendra K. , Gupta Rani , Davidson W. Sheba
Published: October 2023

Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q‐Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41±1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15–90 °C) and was highly thermostable, retaining 100% activity at 60 °C for 24 h. It showed good pH tolerance (3.0–12.0) and was stable over a pH range of 4.0–10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non‐ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2‐mercaptoethanol and potassium ferrocyanide. At a concentration of 100 μM, 3,4‐dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p‐nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze‐drying and had a shelf‐life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3‐regiospecificity and was stable in various organic solvents.
Production of cholesterol oxidase by Rhodococcus equi No. 23 in a jar fermenter
Authors: Chou Cheng‐Chun , Lee Ming‐Tsung , Chen Wen‐Chang
Published: October 2023

Rhodococcus equi No. 23 was grown in a batch fermenter. The effects of cultivation temperature, pH of the culture medium, aeration rate and agitation speed on the production of cholesterol oxidase (CholOx) by the test organism were examined. Results revealed that the cultivation temperature, the pH of the medium, the aeration rate and the agitation speed all affected the production of CholOx by R. equi No. 23. Adjusting the operation variables during the cultivation period increased the production of CholOx effectively and prevented the occurrence of overflow of foam during the fermentation period. A maximum CholOx activity of 0.34 unit/ml with a volumetric production rate of 0.011 unit/h per ml could be achieved in 30 h of cultivation at an aeration rate of 5.0 l/min, if the pH of the culture medium, the cultivation temperature and the agitation speed were controlled at 6.5, 39 °C and 200 rev./min respectively during the first 24 h of cultivation, then shifted to 7.5, 37 °C and 300 rev./min respectively.
Enzymic acylation of 506U78 (2‐amino‐9‐β‐D‐arabinofuranosyl‐6‐methoxy‐9H‐purine), a powerful new anti‐leukaemic agent
Authors: Mahmoudian Mahmoud , Eaddy John , Dawson Mike
Published: October 2023

A practical enzymic approach for acylation of 506U78 (2‐amino‐9‐β‐D‐arabinofuranosyl‐6‐methoxy‐9H‐purine), a powerful anti‐leukaemic agent, is described. Novozyme‐435, an immobilized preparation of Candida antarctica lipase, was used to acylate 506U78 regioselectively at the 5′‐position. This rendered the compound more soluble and bioavailable. Vinyl acetate was used as the acyl donor and reactions were carried out in anhydrous 1,4‐dioxane with up to 100 g/l of substrate input. Bioconversions were optimised to achieve impurity (3′‐mono‐ and di‐acetates) levels of less than 0.5%.
Solvent effect on kinetics of appearance of neokyotorphin, VV‐haemorphin‐4 and a bradykinin‐potentiating peptide in the course of peptic hydrolysis of bovine haemoglobin
Authors: Lignot Brigitte , Froidevaux Renato , Nedjar‐Arroume Naïma , Guillochon Didier
Published: October 2023

The kinetics of appearance of bioactive peptides (neokyotorphin, VV‐haemorphin‐4 and a bradykinin‐potentiating peptide) were investigated in the course of the hydrolysis of haemoglobin by pepsin at 23 °C in 0.1 M sodium acetate buffer at pH 4.5. Isolation of these peptides was performed in one step by C4 reversed‐phase HPLC. We have shown that neokyotorphin, and VV‐haemorphin‐4 are two final peptides and that the bradykinin‐potentiating peptide is an intermediate peptide. At pH 4.5, LVV‐haemorphin‐7, VV‐haemorphin‐7 and VV‐haemorphin‐4 appeared successively in the course of the peptic hydrolysis. At pH 2, the optimum pH of the pepsin hydrolysis, VV‐haemorphin‐4 was not detected. The effect of the composition of the solvent on the peptic hydrolysis was studied to improve the preparation of these active peptides. The kinetics of appearance of these peptides were compared in the presence of 20% (v/v) ethanol, a stabilizing solvent of haemoglobin and in the presence of urea, a denaturant agent. The best conditions for preparing neokyotorphin and VV‐haemorphin‐4 were to achieve the peptic reaction in the buffer alone or in the presence of urea at a high degree of hydrolysis. For the preparation of the bradykinin‐potentiating peptide, the hydrolysis of haemoglobin in the urea‐containing buffer at a moderate degree of hydrolysis of 10% was suitable.
Immobilized lipoxygenase in a packed‐bed column bioreactor: continuous oxygenation of linoleic acid1
Authors: Hsu An‐Fei , Wu Emily , Shen Siyuan , Foglia Thomas A. , Jones Kerby
Published: October 2023

The continuous oxygenation of linoleic acid (LA) by immobilized lipoxygenase (LOX) was studied. Enzymatic oxidation was carried out in a recirculating packed column reactor using immobilized LOX as the stationary phase and LA as the substrate. The column, when packed with LOX immobilized in either a calcium alginate sol‐gel matrix or a phyllosilicate sol‐gel matrix, is equivalent to five continuous stirred tank reactors (CSTRs). The reactor cascade was calculated from the residence‐time distribution for the reactor. Based on mass‐balance calculations, a set of mathematical equations for predicting the concentration of oxygenated product generated in each CSTR was calculated. Product formation in the packed column reactor was simulated and results calculated with the model were compared with the experimental results. The data indicated that product yield (hydroperoxyoctadecadienoic acid, HPOD) increased asymptotically with reaction time. Experimentally, when the bioreactor was packed with calcium alginate sol‐gel‐immobilized LOX, an initial linear increase in HPOD production with time was observed, but reached a steady state. For the bioreactor packed with phyllosilicate sol‐gel‐immobilized LOX, initial HPOD production increased more rapidly but reached a lower steady‐state concentration. From these data, a simple computer simulation model was developed to determine the process kinetics of this reactor design.
Purification and characterization of a Pseudomonas sp. lipase and its properties in non‐aqueous media
Authors: Dong Huan , Gao Shujuan , Han Si‐ping , Cao Shu‐gui
Published: October 2023

An extracellular lipase from Pseudomonas sp. was purified to homogeneity by extraction, Bio‐gel P‐10 chromatography and Superose 12B chromatography, and a 37‐fold purification was attained. The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/PAGE electrophoresis indicated a molecular mass of 30 kDa for this lipase. Its isoelectric point was 4.5. The optimum pH and temperature for hydrolysis were 7.0–9.0 and 45–60 °C, respectively. The enzyme was stable between pHs 6 and 12 and below 60 °C. In the presence of Ca2+ and Bi3+, the lipase activity was dramatically enhanced by 250% and 154%, respectively. Fe3+, Fe2+, Al3+, Zn2+ and Mn2+ could inhibit this lipase, but Ag+ and Pb2+ showed no influence on hydrolysis activity. Properties of purified lipase for lactonization in organic solvent were also determined. The purified lipase displayed the characteristic of ‘pH memory’ in organic media. This lipase was also thermostable in organic solvent with an optimum temperature range from 45 to 60 °C. Salt dramatically affected the lactonization activity of this lipase.
Towards molecular farming in the future: using plant‐cell‐suspension cultures as bioreactors
Authors: Fischer Rainer , Emans Neil , Schuster Flora , Hellwig Stephan , Drossard Jürgen
Published: October 2023

Plant‐suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant‐suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on discussing the generation of transformed suspension‐cell lines expressing recombinant proteins, like antibodies, and recombinant‐protein downstream processing and purification.
Performance comparison of Protein A affinity‐chromatography sorbents for purifying recombinant monoclonal antibodies
Authors: Fahrner Robert L. , Whitney Duncan H. , Vanderlaan Martin , Blank Gregory S.
Published: October 2023

We describe the performance characteristics of five Protein A affinity‐chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host‐cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.
Dermal and transdermal delivery of protein pharmaceuticals: lipid‐based delivery systems for interferon α
Authors: Foldvari Marianna , Baca‐Estrada Maria E. , He Zhihong , Hu Jiaping , Attah‐Poku Samuel , King Martin
Published: October 2023

The dermal and transdermal delivery of protein pharmaceuticals faces enormous challenges, and at the same time has very significant potential for the non‐invasive treatment of both localized and systemic diseases. In this article we review the various approaches used to enhance and control the delivery of protein therapeutic agents through the dermal barrier. We show results of the delivery of interferon (IFN) α, an antiviral agent used in the treatment of condylomata acuminata (genital warts), using lipid‐based delivery systems (LBDS). In the general category of LBDS, we investigated the use of liposomes and fatty acylation as ways to increase IFNα delivery into human skin.
Selectivity modification of chymotryptic hydrolysis of haemoglobin by its adsorption on a solid phase
Authors: Kosciarz Elisabeth , Vercaigne‐Marko Dominique , Scellier Natacha , Nedjar‐Arroume Naïma , Guillochon Didier
Published: October 2023

A change of selectivity of the chymotryptic hydrolysis of haemoglobin was evidenced when the protein was adsorbed on to a negatively charged hydrophobic support. The hydrolysis in heterogeneous phase improved the obtaining of positively charged and hydrophobic peptides as carriers of water‐insoluble molecules. Haemoglobin adsorption on Amberlyst 15Wet was carried out in 0.1 M Tris/HCl buffer at pH 6.0. Chymotryptic hydrolysis was performed for 72 h at 37°C in the same buffer. In solution, the presence of SDS was necessary to achieve the complete hydrolysis of haemoglobin chains, whereas it was not needed when haemoglobin was previously adsorbed on to the resin. The hydrolysis proceeded more slowly in heterogeneous phase than in homogeneous solution because of the diffusional restrictions but, at the end of the hydrolysis, the peptide populations were very different, as shown by reversed‐phase HPLC. Moreover their functional properties were different too, since the haemoglobin hydrolysate obtained by heterogeneous catalysis had a better solubilizing ability towards the water‐insoluble molecule, protoporphyrin IX, a photosensitizer for photodynamic therapy. A time‐course study of the hydrolysis was performed to follow the evolution of a marker peptide (1‐14α), which allowed us to explain the change in the selectivity of the chymotryptic reaction. This change could be due to a slowing down of the cut‐off of some sites interacting with the support.
A novel efficient enzyme‐immobilization reaction on NH2 polymers by means of L‐ascorbic acid
Authors: Tiller Jörg , Berlin Peter , Klemm Dieter
Published: October 2023

A new enzyme‐immobilization reaction by means of L‐ascorbic acid (ASA) is described using NH2 polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH2‐polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N‐dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH2‐polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff’s‐base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH2‐polymer film, as suggested by results from investigations of the ASA reaction with n‐butylamine. The immobilized enzymes can be stored at 4 °C in bidistilled water for at least 1 month without becoming detached from the NH2‐polymer film and without diminished enzyme activity. The apparent Km values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the Km value of the immobilized GOD with different NH2 polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.
Evaluation of refolding conditions for a recombinant human interleukin‐3 variant (daniplestim)
Authors: Boyle Denis M. , McKinnie Russell E. , Joy William D. , Violand Bernard N. , McLaughlin Joseph K. , Landis Bryan H.
Published: October 2023

The refolding of daniplestim, a human interleukin‐3 variant (SC‐55494) from Escherichia coli inclusion bodies, was optimized using a reversed‐phase HPLC method developed to permit quantification of the reduced and oxidized forms of daniplestim. The presence of cysteine or dithiothreitol accelerated refolding of daniplestim from E. coli inclusion body slurries dissolved in urea or guanidine solutions and was complete in 4–6 h. Regardless of the dissolution and refolding protocol used to renature daniplestim, equivalently bioactive protein was produced. Under refolding conditions, no covalent modification of daniplestim by cysteine or cyanate was observed. The folding process was characterized further by following the unfolding of purified daniplestim by far‐UV CD and fluorescence spectroscopies under both oxidizing and reducing conditions at pH values between 7 and 11. Formation of the single disulphide bond had a large stabilizing effect on daniplestim structure (≈4–5 kCal at pH 9·5). This thermodynamic stabilization drove the refolding process towards the native form, even under conditions where the reduced protein was largely unfolded. From these data, scaleable refolding conditions for daniplestim were established.
The use of differential scanning calorimetry (DSC) to determine the correctness of folding of cloned proteins
Authors: Brewer John M.
Published: October 2023

The advantages and limitations of use of the differential scanning calorimetry technique to determine whether cloned and expressed mutant proteins are folded correctly are explained and discussed. Examination of stabilization of these proteins to thermal denaturation by specific ligand binding can provide the required information.
WINNERS AND LOSERS ON NYSE: A RE‐EXAMINATION USING DAILY CLOSING BID‐ASK SPREADS
Authors: Akhigbe Aigbe , Gosnell Thomas , Harikumar T.
Published: October 2023

We study a sample of NYSE stocks that experienced a large one‐day price change during 1992 and were reported as daily largest percentage gainers and largest percentage losers in the Wall Street Journal. The sample indicates significant reversals during the immediate post‐announcement period. We test for market efficiency by using bid‐ask spreads obtained from the transactions data for the days immediately after the announcement. The overall results indicate that the returns during the reversal period are less than the average bid‐ask spread during the same time. We also find that major losers, firms with −20 percent to −50 percent event‐date abnormal returns, experience price reversals generating returns that are significantly greater than the average bid‐ask spread during that period. We interpret this result as consistent with the overreaction hypothesis. A test of a trading rule to exploit this overreaction is not profitable, providing support for weak‐form market efficiency.