bluuebunny/crossref_metadata_2025 huggingface dataset:
Dapagliflozin pharmacokinetics is similar between patients with heart failure with reduced ejection fraction and patients with type 2 diabetes mellitus
Authors: Melin Johanna , Parkinson Joanna , Hamrén Bengt , Penland Robert C. , Boulton David W. , Tang Weifeng
Published: October 2023

Dapagliflozin was recently approved for use in adults with chronic heart failure with reduced ejection fraction (HFrEF) with/without type 2 diabetes mellitus (T2DM). The objectives of this analysis were to characterize dapagliflozin pharmacokinetics in patients with HFrEF and to compare dapagliflozin systemic exposure between adults with HFrEF with/without T2DM and adults with T2DM. A nonlinear mixed‐effects modelling approach was applied; the population‐pharmacokinetic model was developed using 9735 dapagliflozin plasma concentrations from 2744 patients. The final two‐compartmental model adequately described the observed dapagliflozin concentrations, with a similar estimated apparent clearance compared with a previous estimate in patients with T2DM without HF and in healthy subjects (23.0 [95% CI: 22.6–23.9] L/h vs. 22.9 [95% CI: 22.1–23.7] L/h). The model‐predicted median area under the dapagliflozin concentration–time profile was ≤1.2‐fold higher in patients with HFrEF vs. those with T2DM without HFrEF, which is not considered clinically relevant. Dapagliflozin exposure was similar between patients with HFrEF with/without T2DM and T2DM patients without HFrEF.
Low‐impact laparoscopy in colorectal resection—A multicentric randomised trial comparing low‐pressure pneumoperitoneum plus microsurgery versus low‐pressure pneumoperitoneum alone: The PAROS II trial
Authors: Marichez Arthur , Eude Audrey , Martenot Mathieu , Celerier Bertrand , Capdepont Maylis , Rullier Eric , Denost Quentin , Fernandez Benjamin
Published: October 2023

Introduction

Low‐pressure pneumoperitoneum (LLP) in laparoscopy colorectal surgery (CS) has resulted in reduced hospital stay and lower analgesic consumption. Microsurgery (MS) in CS is a technique that has a significant impact with respect to postoperative pain. The combination of MS plus LLP, known as low‐impact laparoscopy (LIL), has never been applied in CS. Therefore, this trial will assess the efficacy of LLP plus MS versus LLP alone in terms of decreasing postoperative pain 24 h after surgery, without taking opioids.

Method

PAROS II will be a prospective, multicentre, outcome assessor‐blinded, randomised controlled phase III clinical trial that compares LLP plus MS versus LLP alone in patients undergoing laparoscopic surgery for colonic or upper rectal cancer or benign pathology. The primary outcome will be the number of patients with postoperative pain 24 h after the surgery, as defined by a visual analogue scale rating ≤3 and without taking opioids. Overall, PAROS II aims to recruit 148 patients for 50% of patients to reach the primary outcome in the LLP plus MS arm, with 80% power and an 5% alpha risk.

Conclusion

The PAROS II trial will be the first phase III trial to investigate the impact of LIL, including LLP plus MS, in laparoscopic CS. The results may improve the postoperative recovery experience and decrease opioid consumption after laparoscopic CS.
Regional oceanography affects humpback whale entanglements in set fishing gear
Authors: Schilling Hayden T. , Dedden Adelaide V. , Crocetti Susan , Liggins Geoff , Lorigan Shona , Marshall Andrew , Rogers Tracey L. , Schaeffer Amandine , Suthers Iain M. , Johnson Daniel D.
Published: October 2023

Humpback whales Megaptera novaengliae undertake annual migrations along eastern Australia, where they can be susceptible to entanglement in set fishing gear. This can potentially result in both sublethal and fatal outcomes for the whales, and financial losses for the fishing industry, including loss of gear and catch. Interannual fluctuations in entanglement records suggest that dynamic oceanographic features may be associated with the whales' migration path, and therefore, risk of entanglement. Using records of demersal fish traps off the coast of New South Wales (NSW), we identify two areas of high fishing pressure and entanglement risk overlap. Within each area, we combine entanglement observations with metrics associated with the East Australian Current (EAC) and climate (i.e., southern oscillation index). We show that the strength and position of the EAC was associated with the likelihood of entanglement in set fishing gear. In northern NSW (30.3° S), entanglement risk was highest between July and October when the EAC is relatively close to the coast. In contrast, there was no evidence suggesting that oceanography further south in the separation zone of the EAC (33.8° S) was associated with risk of entanglement. In the years following a strongly negative SOI (i.e., El Niño events), entanglement risk was also reduced. While we provide clear linkages with regional oceanographic dynamics and entanglement risk off NSW, if a dynamic ocean management approach to reducing entanglements is to be realized, further research into the fine‐scale movements of humpback whales is needed.
The utilization of molecular microscope in management of heart transplant recipients in the era of noninvasive monitoring
Authors: Mehlman Yonatan , Valledor Andrea Fernandez , Moeller Cathrine , Rubinstein Gal , Lotan Dor , Rahman Salwa , Oh Kyung T. , Bae David , DeFilippis Ersilia M. , Lin Edward F. , Lee Sun Hi , Raikhelkar Jayant K. , Fried Justin , Theodoropoulos Kleanthis , Colombo Paolo C. , Yuzefpolskaya Melana , Latif Farhana , Clerkin Kevin J. , Sayer Gabriel T. , Uriel Nir
Published: October 2023

Introduction

Monitoring for graft rejection is a fundamental tenet of post‐transplant follow‐up. In heart transplantation (HT) in particular, rejection has been traditionally assessed with endomyocardial biopsy (EMB). EMB has potential complications and noted limitations, including interobserver variability in interpretation. Additional tests, such as basic cardiac biomarkers, cardiac imaging, gene expression profiling (GEP) scores, donor‐derived cell‐free DNA (dd‐cfDNA) and the novel molecular microscope diagnostic system (MMDx) have become critical tools in rejection surveillance beyond standard EMB.

Methods

This paper describes an illustrative case followed by a review of MMDx within the context of other noninvasive screening modalities for rejection.

Conclusions

We suggest MMDx be used to assist with early detection of rejection in cases of discordance between EMB and other noninvasive studies.
A report of a prospective randomized trial of extended‐release tacrolimus versus immediate release tacrolimus after liver transplantation with anti‐thymocyte induction in a steroid free protocol
Authors: Helmick Ryan A. , Eymard Corey M. , Naik Surabhi , Eason James D. , Nezakatgoo Nosratollah , Nair Satheesh , Vanatta Jason M.
Published: October 2023

Purpose

Our study hypothesis was that once daily dosing of extended‐release tacrolimus (XRT) would be a safe and effective immunosuppression (IS) with the potential to decrease adverse events (AEs) associated with immediate release tacrolimus (IRT) after liver transplantation (LT).

Methods

All patients receiving LT at our center received rabbit anti‐thymocyte globulin (RATG) induction therapy. Eligible patients were randomized in a 1:1 fashion to receive either XRT or IRT. Antimicrobial prophylaxis was the same between arms, and both groups received an antimetabolite for the first 6 months following LT. Patients were then followed at pre‐determined study intervals for the following year after LT. We administered the RAND‐36SF survey to assess patient's health‐related quality of life at pre‐determined intervals. All analysis was performed with an intention to treat basis.

Results

We screened 194 consecutive patients and enrolled 110. Our control and study arms were well matched. Transplant characteristics were similar between groups. At all timepoints, both arms had similar serum creatinine and estimated glomerular filtration rate (eGFR), calculated by MDRD6 equation, with post‐transplant GFRs between 60 and 70 mL/min/1.73 m2. Tacrolimus trough levels were similar between arms. The XRT arm had fewer AEs (166) and fewer serious AEs (70) compared to IRT (201 and 99, respectively). AEs most commonly were renal, infectious, or gastrointestinal in nature. While not statistically significant, XRT was held temporarily (25 vs. 35 cases) or discontinued (3 vs. 11 cases) less frequently than IRT and had fewer instances of rejection (7 vs. 12 cases).

Conclusion

This analysis showed that XRT is safe and effective as de novo maintenance IS in a steroid‐free protocol with RATG.
Time preference and economic growth: The case for China and international comparisons
Authors: Sun Lixin
Published: October 2023

In this paper, first, we measure Chinese time preferences as a whole and by region based on the Euler equation under an assumption of rational expectation. The calculating results show that the rates of Chinese time preference are markedly low for most hypothetical risk attitudes over the concerning period, and there exists heterogeneity in time preferences across regions in China. Second, compared with the estimates for other advanced and emerging countries under the same assumptions and same methodology, the rates of Chinese time preference are the lowest for any given risk preference, suggesting that Chinese are more patient during the time of our examinations. Third, our measured time preferences are powerful in predicting China's high saving rates and rapid growth rates of gross domestic product. In addition, the differences in economic growth rates between China and other sample countries can be partially explained by the differences of estimated cross‐countries time preferences within our sample period. Fourth, we investigate the additional factors that affect the Chinese time preferences. Our study provides insights on the regional and international differences in economic growth rates and other economic outcomes.
Comparative study of solid‐state fermentation with Lactobacillus plantarum versus Saccharomycopsis fibuligera on anti‐nutritional factors, nutritional values and antioxidant activity of faba bean (Vicia faba L.) meal
Authors: Xu Ruiping , Han Jinquan , Tian Tie , Zhu Nengbin , Chen Yiling , Wangkahart Eakapol , Zhang Zhiqi , Liu Jun , Yu Denghang , Wei Jin , Xu Hongsen
Published: October 2023

Solid‐state fermentation (SSF) is widely used to improve the utility value in the food and feed industries. In the present research, Lactobacillus plantarum and Saccharomycopsis fibuligera were used for SSF, and then, the nutritional values, anti‐nutritional factors (ANFs) and antioxidant activities of faba bean meal (FBM) were comparatively analysed. The results revealed that SSF with S. fibuligera and L. plantarum effectively reduce the tannin and phytic acid contents and improve the nutritional quality and antioxidant capacity in FBM. Meanwhile, the effects of SSF with L. plantarum on degrading tannin and increasing titratable acidity, trichloroacetic acid‐soluble protein and in vitro protein digestibility were better treated with S. fibuligera. However, antioxidant activities and the amount of phenol and flavonoid were improved more in FBM treated with S. fibuligera than those fermented with L. plantarum. Taken together, the results indicated that both L. plantarum and S. fibuligera could be used for SSF of FBM, and SSF with those two microorganisms has great potential in improving the nutritional values, antioxidant capacities and decreasing ANFs contents of FBM.
Divide and conquer: Relating patent quality and value in a conceptual framework based on a systematic review
Authors: Ananthraman Srinivasan , Cambré Bart , Kittler Markus , Delcamp Henry
Published: October 2023

Patents as intangible assets are subjects of burgeoning empirical research. However, there is limited knowledge of how patent quality and patent value can be conceptualized, distinguished, and related. Distinguishing these concepts and relating them in a theoretical framework would enable the assessment and improvement of patent quality, which has implications for all the stakeholders in patents. We ground this study in the emergent ex‐ante theory of patent value and conduct a systematic review of 340 papers that investigate patent quality or value. Based on a comparative analysis of the patentability standards adopted by the patent offices in the United States, Europe, and Japan, we delineate four dimensions of patent quality—subject matter, utility, non‐obviousness or inventive step, and sufficiency of disclosure. Our study contributes to theory by providing an elaborated conceptual model that relates the different dimensions of patent quality and patent value and mapping the different types of indicators of patent quality and value onto the corresponding patent quality or value dimensions. Our study suggests that patent policymakers can incentivize innovators to file patent applications of high quality, which would reduce the incidence of poor‐quality patents in the system and improve the efficiency and reputation of the patent office.
Visual audience gatekeeping on social media platforms: A critical investigation on visual information diffusion before and during the Russo–Ukrainian War
Authors: Durani Khalid , Eckhardt Andreas , Durani Walid , Kollmer Tim , Augustin Nils
Published: October 2023

Gatekeepers, including prerogative elites and institutions, possess multifaceted tools for controlling information. Historically, these gatekeepers have systematically instrumentalized visual information to shape audience perceptions in alignment with specific agendas. However, the advent of social media platforms has disrupted these power dynamics, empowering audiences through user‐driven visual information exchanges. This has led to visual audience gatekeeping whereby users act as audience gatekeepers by selectively disseminating visual content, thus shaping information diffusion. While this democratisation of gatekeeping holds considerable promise for user empowerment, it also presents risks. These risks stem from the inherently persuasive yet manipulative qualities of visual content, compounded by users' susceptibility to unconscious biases, especially during polarising events. Therefore, we critically investigated visual audience gatekeeping in the context of the Russo–Ukrainian War, specifically focusing on a Reddit subforum devoted to Russia‐related discussions. We find that visual audience gatekeeping operates within a paradigm that reflects the social reality the audience embraces. Audience gatekeepers disseminate visual information both to reproduce and defend an idealised conception of this social reality. Collectively, they produce a visual echo chamber characterised by low information diversity, which, in turn, reinforces and perpetuates the incumbent paradigm. However, heightened social tensions can trigger a paradigm shift and amplify the reproduction and defence processes, increasing the potential for the diffusion of more radical and extreme visual narratives. Based on our findings, we propose a theoretical model of visual audience gatekeeping, which has important implications for research on visuals, gatekeeping and social media platforms.
Epithelioid Leiomyosarcoma of the Skin
Authors: Suh Kee‐Suk , Jang Min‐Soo , Chae Young‐Soo , Kim Sang‐Tae
Published: October 2023

We report a case of epithelioid leiomyosarcoma that developed on the nose of a 55‐year‐old Korean male over a one year period. The lesion was a pea sized, firm, erythematous, painless nodule with erosion in the center. Histologic examination revealed short spindled cells with blunt‐ended nuclei and pleomorphic round to oval epithelioid cells with abundant eosinophilic cytoplasm that were perivascular and densely packed in the dermis. Immunostaining for desmin was negative, although stains for vimentin and smooth muscle actin were both strongly positive.
Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix‐assisted laser desorption ionization‐time‐of‐flight MS
Authors: Klyushnichenko Vadim , Rodenbrock Anja , Thömmes Jörg , Kula Maria‐Regina , Heine Holger , Biselli Manfred
Published: October 2023

SDS/gel capillary electrophoresis (SDS/gel CE) and matrix‐assisted laser desorption ionization‐time‐of‐flight MS (MALDI‐TOF‐MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off‐line coupling of CZE and MALDI‐TOF‐MS for the analysis of biotechnological processes is demonstrated.
Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis
Authors: Guillén Gerardo , Alvarez Anabel , Silva Ricardo , Morera Vivian , González Sonia , Musacchio Alexis , Besada Vladimir , Coizeau Edelgis , Caballero Evelin , Nazabal Consuelo , Carmenate Tania , González Luis J. , Estrada Regla , Támbara Yanet , Padrón Gabriel , Herrera Luis
Published: October 2023

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.
Carboxy‐group modification: high‐temperature activation of charge‐neutralized and charge‐reversed β‐glucosidases from Aspergillus niger
Authors: Rashid Muhammad Hamid , Siddiqui Khawar Sohail
Published: October 2023

Purified β‐glucosidase from Aspergillus niger NIAB280 was chemically modified by 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl)‐carbodi‐imide (EDC) in the presence of glycinamide (GAM) as nucleophile under various conditions to study the role of carboxy groups in the catalytic mechanism of this enzyme. β‐Glucosidase was inactivated by the binding of one mol of EDC per mol of the enzyme with a second‐order rate constant of 4.77 × 10−2 mM min−1. Glucose, as competitive inhibitor, partly protected the active‐site carboxy group against chemical modification, with a Kd of 3.64 mM. The pH dependence of chemical modification by EDC showed that first‐order rate constants decreased with increasing pH, indicating that the proton donating group is a carboxy group. The pKa values of the acidic and basic limbs of the native enzyme were 2.9 and 6.5 respectively. β‐Glucosidase was modified by EDC in the presence of GAM and ethylenediamine dihydrochloride (EDAM) as nucleophiles for 60 min. The effects of neutralization (GAM) and reversal (EDAM) of the negative charges of surface carboxy groups on the kinetic properties of the enzyme were also studied. Native β‐glucosidase, GAM and EDAM had Vmax/Km values of 0.73, 1.22 and 0.60 respectively at 40°C. Interestingly, the activation energy profiles of native β‐glucosidase (103 and 79 kJ/mol) were biphasic, whereas those of GAM (137, 101 and 30 kJ/mol) and EDAM (285, 100 and 29 kJ/mol) were triphasic, indicating significant activation of modified β‐glucosidases at temperatures higher than 50°C. The pKa values of both the active‐site carboxy groups as well as the pH optima of GAM and EDAM were also significantly decreased compared with those of the native β‐glucosidase.
Molecular mass determination for prostate‐specific antigen and α1‐antichymotrypsin complexed in vitro
Authors: Bedzyk William D. , Larsen Barbara , Gutteridge Steve , Ballas Robert A.
Published: October 2023

The results from molecular mass determinations and amino acid analyses for prostate‐specific antigen (PSA), α1‐antichymotrypsin (ACT) and PSA‐ACT complexed in vitro are reported. Molecular masses for two separate PSA‐ACT lots were determined by matrix‐assisted laser desorption ionization mass spectroscopy (MALDI‐MS) coupled to a time‐of‐flight (TOF) detector. Interestingly, both PSA‐ACT lots contained two predominant protein species: 78095± 138 Da (approx. 67% of total protein) and 82519± 104 Da. Because a heterogeneous population was observed and the masses were less than expected on the basis of less sensitive techniques, molecular masses for the individual PSA and ACT components were determined. As expected, PSA possessed a single molecular mass (27755.8 Da). Each ACT raw material lot, however, also contained two predominant species: 55106± 111 and 51414± 32 Da. To assess the amino acid composition, each PSA, ACT and PSA‐ACT lot was subjected to acid hydrolysis for 24, 48 and 72 h followed by amino acid analysis. Experimental results, expressed as mole percentages for each measurable residue and the number of residues per mole of protein, were compared with both the predicted and previously published values and were as expected. These more accurate molecular mass values for ACT and purified PSA‐ACT complex should be considered in preparing and characterizing international standard preparations.
Erythrocyte engineering for drug delivery and targeting
Authors: Magnani Mauro , Rossi Luigia , D'ascenzo Marcello , Panzani Ivo , Bigi Leonardo , Zanella Andrea
Published: October 2023

A new procedure for the encapsulation of non‐diffusible drugs into human erythrocytes was developed. With as little as 50 ml of blood and by using a new apparatus, it was possible to encapsulate a variety of biologically active compounds into erythrocytes in 2 h at room temperature and under blood‐banking conditions. The process, which is based on two sequential hypotonic dilutions of washed red cells followed by concentration with a haemofilter and resealing of red cells, allows a 35–50% cell recovery and approx. 30% encapsulation of added drugs. The resulting processed erythrocytes have a normal survival in vivo and can be modified further, with the same apparatus, to increase their recognition by tissue macrophages to perform as a drug‐targeting system. The new equipment designed and built for this procedure was named ‘Red Cell Loader’.
Investigation of biosynthesis of human lymphotoxin in cells of recombinant Escherichia coli strain
Authors: Denisov Alexander A. , Nikolaeva Olga G.
Published: October 2023

The effect of cultivation conditions on the biosynthesis of human lymphotoxin in recombinant Escherichia coli SG20050/pLT21 strain was studied. Cells of the producing strain were grown in Luria broth containing chloramphenicol. The highest biomass yield of the recombinant strain and plasmid DNA stability were observed under these conditions. To enhance the level of lymphotoxin production an inoculate containing freshly obtained or frozen with glycerol transformants of the producing strain were used, and the cultivation process was performed at 32 °C. As a result, lymphotoxin was synthesized in a soluble form without the formation of inclusion bodies. A study of the protein synthesis dynamics during the cultivation of E. coli at 32 °C showed that the highest lymphotoxin activity was observed during the exponential growth phase, being maximal at the end of the exponential phase and at the beginning of the stationary phase. The set of indicated methods allowed us to maximize and stabilize the production of lymphotoxin in a biologically active form with a final yield of 18–20% from cell protein.
Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level
Authors: Dellinger Marc , Geze Marc , Santus René , Kohen Elli , Kohen Cahide , Hirschberg Joseph G. , Monti Marco
Published: October 2023

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring. The mounting as a detector of a cooled charge‐coupled device camera or alternatively of a non‐cooled camera in conjunction with an image intensifier or an investigator (i.e. frame scan accumulator) to enhance sensitivity makes possible the detection of the low‐quantum‐yield NAD(P)H fluorescence at a level comparable to images previously obtained with high‐quantum‐yield fluorochromes. The modulation of mitochondrial autofluorescence by rotenone, carbonyl cyanide p‐ trifluoromethoxyphenylhydrazone and oligomycin, and of cytoplasmic and nuclear autofluorescence by glucose and iodacetamide in CV‐1 kidney epithelial cells, Ehrlich‐Lettre hypotetraploid CCL77 cells and Saccharomyces cerevisiae, provides examples of the usefulness of fluorescence imaging in the study of biopharmaceuticals. The method goes beyond NAD(P)H to the multiplicity of extrinsic and intrinsic probes already available or in development.
Peroxidase production in vitro by Armoracia lapathifolia (horseradish)‐transformed root cultures: effect of elicitation on level and profile of isoenzymes
Authors: Flocco Cecilia Gabriela , Alvarez María Alejandra , María Ana
Published: October 2023

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field‐grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.
O‐Mannosylation of Pichia pastoris cellular and recombinant proteins
Authors: Duman Joseph G. , Miele Robert G. , Liang Hong , Grella Davida K. , Sim Kim Lee , Castellino Francis J. , Bretthauer Roger K.
Published: October 2023

O‐linked saccharides were released from a major cell wall glycoprotein and from cellular mannan–protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (α1‐2)‐linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1–4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O‐ and N‐linked saccharides. Only a very small quantity of N‐linked oligosaccharides was present on the Asn289 consensus site. The major products were O‐linked (α1‐2)‐ linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O‐linked saccharides of mannose containing (α1‐2) glycosidic linkages are present in P. pastoris cells and expressed proteins.
Characterization of soybean lipoxygenase immobilized in cross‐ linked phyllosilicates
Authors: Hsu An‐Fei , Shen Siyuan , Wu Emily , Foglia Thomas A.
Published: October 2023

Lipoxygenase (LOX) is an enzyme that regioselectively introduces the hydroperoxide functionality into polyunsaturated fatty acids, such as linoleic acid (LA). Hydroperoxide derivatives of polyunsaturated fatty acids are of interest because they can serve as important intermediates in the synthesis of chemical and pharmaceutical compounds. In this study, LOX was immobilized in dispersed phyllosilicate layers that were cross‐linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicates. The effects of substrate concentration, reaction temperature and solvent participation were studied on the oxidation of LA by LOX. The temperature optimum for the oxidation of LA by immobilized LOX was 25 °C and values of Km and Vmax for this reaction were 1.7 mM and 0.023 μmol/min respectively. Enzymic activity was stimulated by the addition of 10% (v/v) iso‐octane to the reaction mixture. The immobilized LOX preparation showed a degree of substrate preference that demonstrated that 1,3‐dilinolein was a better substrate than LA in the oxidation reaction, followed in order by 1‐ monolinolein, methyl oleate and trilinolein. In general, LOX immobilized in cross‐linked phyllosilicates retained the physical and chemical characteristics of free LOX.
Chromatographic removal and heat inactivation of hepatitis A virus during manufacture of human albumin
Authors: Adcock Wayne L. , MacGregor Andrew , Davies Jeff R. , Hattarki Meghan , Anderson David A. , Goss Neil H.
Published: October 2023

CSL Limited, an Australian biopharmaceutical company, has recently converted its method of manufacture for human albumin from a traditional Cohn‐ethanol fractionation method to a method employing chromatographic techniques. Studies were undertaken to determine the efficiency of the chromatographic and pasteurization steps used in the manufacture of Albumex® (CSL's trade name for albumin) in removing and inactivating the potential viral contaminant, hepatitis A virus (HAV). The manufacturing process for Albumex® includes three chromatographic steps, two of which are ion‐exchange steps (DEAE‐ Sepharose® Fast Flow and CM‐Sepharose® Fast Flow) and the third is a gel‐filtration step (Sephacryl® S200 HR). The final stage of the Albumex® process involves a bulk pasteurization step where product is held at 60 °C for 10 h. HAV partitioning experiments on the DEAE‐Sepharose® FF and CM‐Sepharose® FF ion‐ exchange and Sephacryl® S200 HR gel‐filtration columns were performed with scaled‐down models of the production‐scale chromatographic Albumex® process. Production samples collected before each of the chromatographic steps were spiked with HAV and processed through each of the scaled‐down chromatographic columns. Samples collected during processing were assayed and the log10 reduction factors calculated. Inactivation kinetics of HAV were examined during the pasteurization of Albumex® 5 and 20 [5% and 20% (w/v) albumin solutions] held at 60 °C for 10 h. Log10 reductions for HAV through the DEAE‐Sepharose® FF, CM‐ Sepharose® FF and Sephacryl® S200 HR chromatographic columns were 5.3, 1.5 and 4.2 respectively, whereas a 4.4 and a greater than 3.9 log10 reduction in HAV in Albumex® 5 and 20 respectively were achieved during pasteurization.
Development of Bacillus thuringiensis fermentation and process control from a practical perspective
Authors: Yang Xiao‐Ming , Wang Shaw S.
Published: October 2023

Bacillus thuringiensis (Bt) is the most widely used biopesticide producer in the biological control market. It is very critical for the Bt pesticide industry to be able to achieve a high yield in the Bt fermentation process in order to reduce its cost and compete with chemical pesticides in the market. We review the overall development of Bt fermentation process research and provide our point of view for the future research opportunities and potential improvements. This minireview covers the areas of fermentation physiology, growth dynamics and high‐yield process control. It is pointed out that many studies aimed to improve spore count and process research focusing on toxin protein yield is lacking. In addition, significant development opportunities reside in the process development for the genetically engineered Bt strains expressing multiple toxin proteins.
Degradation of human cardiac troponin I after myocardial infarction
Authors: Morjana Nihmat A.
Published: October 2023

Cardiac troponin I (TnI) is the inhibitory subunit of the troponin complex and a specific biochemical marker for myocardial infarction (MI). It is released into the bloodstream within 4–6 h following MI, peaks after 18–24 h and remains elevated for up to 7 days. In this work, I have identified TnI forms present in MI‐patient serum. By immobilizing anti‐TnI antibodies, which recognize various epitopic sites on the TnI molecule, we were able to isolate TnI from MI‐patient serum pools. Western‐blot analysis following SDS/PAGE shows two major TnI fragments with apparent molecular masses of 18000 and 14000 Da. Either or both fragments are seen in serum obtained from individuals with MI. The fragments are generated as a result of proteolytic processing from the C‐terminal region of TnI. Partial processing from the N‐terminal of TnI is also seen and is associated with the generation of the 14000 Da fragment. Very little unprocessed intact TnI is detected in patient serum after MI. The degradation in vitro of cardiac TnI was studied by incubating either bovine or human recombinant TnI in serum. Western‐blot analyses with TnI antibody showed that purified TnI spiked into normal human serum or MI‐patient serum depleted of TnI degrades rapidly to lower molecular mass fragments. Degradation of TnI is associated with a loss in immunological activity. Serum TnI isolated by anti‐TnI antibody, under non‐dissociating conditions, is associated with at least troponin C (TnC) and troponin T (TnT). This complex is bound by anti‐TnI, anti‐TnC and anti‐TnT antibodies.
Immobilization of α‐amylase on poly(vinyl alcohol)‐coated perfluoropolymer supports for use in enzyme reactors
Authors: Yang Yuhui , Chase Howard A.
Published: October 2023

The suitability and potential for the use of poly(vinyl alcohol) (PVA)‐coated solid perfluoropolymers in immobilized‐enzyme engineering have been evaluated by using α‐amylase from Bacillus licheniformis for the hydrolysis of starch. α‐Amylase was covalently immobilized on PVA‐coated poly(tetrafluoroethylene‐hexafluoropropylene) (PVA‐FEP) by covalent coupling with the use of p‐β‐sulphate‐(ethyl sulphonide)‐aniline, 2,4,6‐trichloro‐1,3,5‐triazine, 1.1′‐carbonyldi‐imidazole and 2,2,2‐trifluoroethanesulphonyl chloride activation procedures, and also for comparison with cyanogen bromide‐activated Sepharose 4B. In all cases, immobilization greatly improved the thermostability of the α‐amylase and its resistance to inactivation by 6 M urea. Also the enhancements of enzymic activities with increased temperature were higher for the immobilized enzymes than for the soluble enzyme, and the immobilized α‐amylases were well suited to the continuous hydrolysis of starch conducted at elevated temperatures. Although the specific activities of the enzymes immobilized on PVA‐FEP were lower than for that immobilized to Sepharose 4B, these novel supports showed far superior strength. The enzymes immobilized on PVA‐FEP were able to be readily recovered from stirred batch bioreactors for repeated reuse, whereas the enzymes immobilized to Sepharose were fractured and fragmented under similar conditions of stirring. A conventional fixed‐bed bioreactor was found to be unsuitable for continuous starch hydrolysis owing to an unacceptable build‐up of pressure drop across the bed. However, an expanded bed reactor containing α‐amylase immobilized on solid PVA‐coated perfluorocarbon showed great potential for the continuous hydrolysis of starch. Only 20% of the enzyme activity was lost after use for 3 weeks at 72 °C. It is concluded that PVA‐coated solid perfluorocarbon is a highly promising support for use in immobilized enzyme engineering.
Expression of recombinant Lym‐1 single‐chain Fv in Escherichia coli
Authors: Bin Song Kyung , Won Misun , Meares Claude F.
Published: October 2023

Lym‐1 single‐chain Fv (sFv) can be used for targeted radiodiagnosis and therapy of B‐lymphocytic malignancies. Lym‐1 sFv was constructed and expressed as a glutathione S‐transferase fusion protein, using a (G4S)3 linker connecting the C‐terminus of the VH domain and the N‐terminus of the VL domain of Lym‐1. Six histidine residues and an E Tag epitope were introduced at the C‐terminus of the sFv. Lym‐1 sFv was purified with glutathione–Sepharose 4B affinity chromatography followed by digestion with thrombin. Lym‐1 sFv of 28 kDa was confirmed by Western blotting with anti‐(E Tag) monoclonal antibody. An antigen binding assay of Lym‐1 and a CD study indicated that it is functionally active.
Carbodi‐imide coupling of enzymes to the reversibly soluble insoluble polymer Eudragit S‐100
Authors: Tyagi Renu , Roy Ipsita , Agarwal Ritu , Gupta Munishwar N.
Published: October 2023

The coupling of proteins and enzymes to soluble–insoluble polymers by carbodi‐imide can be performed by using numerous variations of the protocol. This protocol has been investigated for the coupling of five different enzymes, namely wheatgerm acid phosphatase, β‐glucosidase, β‐galactosidase, trypsin and xylanase, to an enteric methacrylate polymer Eudragit S‐100. The following results were found. (1) The activity of the bioconjugate was critically dependent on the physical state of the polymer and the pH of the coupling reaction. For example, in the case of wheatgerm acid phosphatase, the activity of the bioconjugate was 49% when coupling was performed at pH 7·2 and 67% when coupling was performed at pH 4·5. With β‐galactosidase the corresponding values were 57% and 23% and with β‐glucosidase they were 57% and 52% respectively. (2) In some cases, such as β‐glucosidase and β‐galactosidase, it might be necessary to remove excess carbodi‐imide before the addition of the enzyme to the activated matrix. (3) In most of the cases investigated, a sig‐nificant amount of the enzyme (more than 90%) could be bound to the matrix merely by adsorption. (4) More importantly, after the carbodi‐imide coupling procedure, a sufficient fraction of the bound enzyme could be eluted off the matrix, indicating that this was merely adsorbed and not covalently coupled.
An improved, inexpensive method for the large‐scale purification of human nerve growth factor
Authors: Li Suqin , Li Faqing , Tan Weiguo , Yang Nan , Jin Huiying , Chen Huabiao
Published: October 2023

Human nerve growth factor (hNGF) was purified to near homogeneity on a large scale from human term placenta with an improved and inexpensive method. The purification procedure included tissue homogenization, ultrafiltration and single CM‐cellulose column chromatography. The purified hNGF was a 14·4‐kDa protein with an isoelectric point of approximately 9·3. The specific activity of the purified hNGF was approximately 38000 units/mg, and the activity was completely inhibited by the monoclonal antibody against recombinant hNGF (rhNGF). Western‐blot analysis showed that the purified hNGF could interact with the monoclonal antibody against rhNGF.
Generation of a highly immunogenic recombinant enolase of the human opportunistic pathogen Candida albicans
Authors: Sandini Silvia , Melchionna Roberta , Arancia Silvia , Gomez Maria Jesus , La Valle Roberto
Published: October 2023

Enolase, a 46 kDa glycolytic enzyme, is an immunodominant antigen of Candida albicans, an important human opportunistic pathogen. The full‐length coding sequence of C. albicans enolase gene was subcloned into the prokaryotic expression vector pDS56/RBSII, His6/E− under the control of an inducible promoter to produce a His6‐tagged enolase. The recombinant protein was purified to homogeneity by one‐step nickel‐chelate affinity chromatography. It was recognized by a monoclonal antibody specific for C. albicans enolase, as well as by anti‐enolase antibodies present in human sera (IgG). The recombinant protein promptly elicited antibody in mice and detected immune responses in normal human subjects that were comparable to those generated by the native C. albicans enolase. Thus this new recombinant enolase constitutes a valuable reagent for studying the possible role of this protein in anti‐Candida immune response.
Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus
Authors: Mori Shuji , Okamoto Motoi , Nishibori Masahiro , Ichimura Mitsuko , Sakiyama Junko , Endo Hiroshi
Published: October 2023

Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 μmol p‐nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p‐nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60–70 °C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N‐terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.
Colorimetric capture assay for human‐immunodeficiency‐virus‐1 reverse transcriptase activity
Authors: Rytting Ann‐Sofie , Åkerblom Lennart , Gronowitz J. Simon , Källander Clas F.R.
Published: October 2023

The development of a colorimetric capture assay for HIV‐1 reverse transcriptase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all performed in the same microtitre plate. Mouse monoclonal anti‐RT antibodies of subclass G2a were bound by polyclonal goat anti‐(mouse IgG2a) immobilized in the wells of a microtitre plate. The monoclonal antibodies (mAbs) were selected for their ability to bind HIV‐1 RT without hampering the polymerase activity. The assay system first involved the RT's adherence to the immobilized mAbs. Non‐specific enzymes and other impurities were removed by a simple wash, after which an RT reaction mixture containing BrdUTP as nucleotide substrate was added. After the RT reaction substrate and product had been separated by washing of the plate, the amount of BrdUMP‐DNA in the wells was finally detected with alkaline‐phosphatase‐conjugated mouse anti‐BrdU antibodies of subclass IgG1. The background signal in this system was similar to the signals obtained with control wells coated with BSA only. A detection limit of 1.2 μ‐units of RT activity, corresponding to 0.3 pg of RT protein, was obtained for the capture assay when applying colorimetric product detection. The assay detected RTs from HIV‐1 subtypes A and B and one of the two D type isolates tested. None of the five non‐HIV‐1 RTs tested was found positive. At least 50 μl of human serum or plasma per sample could be included in the capture assay without adverse effects on the recovery of the RT activity.
Accumulation of fibrinogen‐coated microparticles at a fibrin(ogen)‐rich inflammatory site
Authors: DeAnglis Ashley P. , Fox Michael D. , Retzinger Gregory S.
Published: October 2023

We have developed methods for coating with fibrinogen both liposomes and microscopic droplets of olive oil. Because the fibrinogen bound to them is functional in the classic sense of fibrin gelation, the coated microparticles may have potential as vehicles for the targeted delivery of various molecules to sites of fibrin(ogen) deposition in vivo. So that we could assess directly this potential, we first established a method for eliciting reproducibly a focal, fibrin(ogen)‐rich, inflammatory lesion in a hind footpad of mice. We then monitored the tissue distribution of fibrinogen‐coated microparticles following their injection into the tail vein of mice bearing this well‐defined lesion. As happens with most microparticles following their intravenous administration, liposomes and oil droplets, whether coated with fibrinogen or not, accumulate rapidly in the liver and spleen of treated animals. Indeed, in the case of oil droplets, accumulation of fibrinogen‐coated microparticles in those organs and in the lungs is even greater than that of fibrinogen‐free microparticles. However, as distinct from fibrinogen‐free liposomes and oil droplets, fibrinogen‐coated microparticles also accumulate in the inflamed hind footpad. We conclude that fibrinogen‐coated liposomes and oil droplets do have potential as vehicles for delivering molecules to sites of fibrin(ogen) deposition in vivo.
Purification and characterization of a levanbiose‐producing levanase from Pseudomonas sp. No. 43
Authors: Jung Kang Eun , Lee Sang Ok , Lee Jae Dong , Lee Tae Ho
Published: October 2023

A levanbiose‐accumulating levanase from Pseudomonas sp. No. 43 was purified to a homogeneous state by (NH4)2SO4 fractionation and by chromatography on DEAE‐Toyopearl 650 M and phenyl‐Toyopearl 650 M columns. The molecular mass and isoelectric point of the enzyme were estimated to be 36 kDa and 5.7 respectively; the optimal pH and temperature for the enzyme reaction were pH 7.0 and 40 °C respectively. The purified enzyme was stable in the pH range 6.0–8.0 at 20 °C and stable up to 50 °C at pH 7.0. The enzyme's activity was inhibited by MnCl2, CoCl2, AlCl3, EDTA and potassium permanganate. The levanase was specific towards the 2,6‐β‐D‐fructosidic linkages of levan and did not hydrolyse other polysaccharides among those examined. The enzyme is an exohydrolase of levan and produced levanbiose as a sole product; the limits of hydrolysis of levans from Zymomonas mobilis and Serratia sp. were 65% and 80% respectively.
Photoproduction of L‐tryptophan from indole and glycine by Rhodobacter sphaeroides OU5
Authors: Rajasekhar N. , Sasikala Ch. , Ramana Ch. V.
Published: October 2023

A purple non‐sulphur anoxygenic phototrophic bacterium Rhodobacter sphaeroides could synthesize L‐tryptophan from indole and glycine with intermediate formation of D,L‐alanine and L‐serine. Presence of externally supplied keto acids has enhanced the rate and yields of L‐tryptophan photoproduction.