Urinary tract infections are common and occasionally life threatening condition amongst diabetic and non-diabetic patients. The present study investigated the distribution of β-lactamase enzyme producing uropathogenic E.coli isolates amongst diabetic and non-diabetic patients. Collected isolates were identified and confirmed as uropathogenic E.coli by standard microbiological procedure and antibiotic susceptibility were screened by Kirby-Bauer disc diffusion method. Phenotypic detection of β- lactamases such as ESBL, AmpC β- lactamase and carbapenemase were determined by double disc diffusion method, disc approximation test and modified Hodge test respectively. β- lactamase encoding genes such as TEM, SHV,CTX-M for ESBLs, ACC, EBC, CIT,DHA, MOX and FOX for AmpC-β-lactamase and KPC, IMP, VIM, NDM and OXA-48 for carbapenemase were detected by PCR method. Out of 306 isolates, 280 (140 from diabetic and 140 from non-diabetic UTI patients) non-repetitive isolates were identified and confirmed as Uropathogenic E.coli. Antibiotic screening revealed that 124 diabetic (88.57%) and 117 non-diabetic (83%) isolates were resistant to at least one antibiotic included in the study. Phenotypic confirmation of various β-lactamase enzymes such as ESBL (diabetic=85/85; 100% and non-diabetic=78/79; 98.7%), AmpC β-lactamase (diabetic=26/36; 72.2% and non-diabetic=18/26; 69%) and carbapenemase (diabetic=19/25; 76% and non-diabetic=13/15; 86.6%) were determined. ESBL encoding genes (diabetic=74/85; 87.05% and non-diabetic=65/78(83.3%), AmpC β- lactamase encoding genes (diabetic=21/26; 80.8% and non-diabetic=12/18; 66.6%) and carbapenemase encoding genes (diabetic=18/19; 94.73% and non-diabetic=11/13; 84.61%) were genotypically confirmed. This study showed that the number of beta-lactamase producing UPEC isolates were phenotypically and genotypically higher in diabetic than non-diabetic patients.

An efficient synergistic trityl cation ([Ph3C][B(C6F5)4])/triflic anhydride (Tf2O) catalyzed alkylation of phenols with alcohols is reported. Benefiting from formation of the triflate in situ, cheap and readily available active alcohols can be used as the alkylating reagents and the reaction proceeds under mild reaction conditions, with broad substrate scope. This protocol enables the synthesis of ortho-selective phenols and 2,4,6-trisubstitued phenols containing three different alkyl groups. tert-Amyl triflate was synthesized and mechanistic studies support a triflate-mediated alkylation process.

Sialic acid is a unique sugar moiety that resides in the distal and most accessible position of the glycans on mammalian cell surface and extracellular glycoproteins and glycolipids. The potential for sialic acid to obscure underlying structures has long been postulated, but the means by which such structural changes affect directly biological processes continues to be elucidated. Here, we appraise the growing body of literature detailing the importance of sialic acid for the generation, differentiation, function, and death of hematopoietic cells. We conclude that sialylation is a critical post-translational modification utilized in hematopoiesis to meet the dynamic needs of the organism by enforcing rapid changes in availability of lineage-specific cell types. Though long thought to be generated only cell-autonomously within the intracellular ER-Golgi secretory apparatus, emerging data also demonstrate previously unexpected diversity in the mechanisms of sialylation. Emphasis is afforded to the mechanism of extrinsic sialylation, whereby extracellular enzymes remodel cell surface and extracellular glycans, supported by charged sugar donor molecules from activated platelets.

Background: The HER2-targeted antibody‒drug conjugate (ADC) is a novel approach for anti-HER2 treatment, and its efficacy in breast cancer patients has been demonstrated in clinical studies. However, the overall efficacy and safety of the various HER2-targeted ADCs in patients with gastric and gastroesophageal junction cancer has not been reported. Method: The PubMed, Embase, Web of Science, Cochrane Central Register of Controlled Trials (CENTRAL) and ClinicalTrials.gov databases were systematically searched. We assessed the quality of the included studies and then/span>extracted the overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) to conduct the meta-analysis. Furthermore, we performed subgroup and sensitivity analyses to explore the sources of heterogeneity. The MINORS and RoB2 were used to assess the quality of the included studies, and STATA 17.0 software was used for data analysis. Results: Six single-arm studies and 2 randomized controlled trials (RCTs) with a total of 871 patients were included. The pooled ORR and DCR were 29% (95% CI: 20%-38%) and 71% (95% CI: 56%-86%), respectively. The pooled mOS and mPFS were 9.68 months (95% CI: 7.78-11.58 months) and 5.60 months (95% CI: 4.59-6.61 months), respectively. The incidence rates of all-grade adverse events (AEs) and grade≥3 AEs were 98.8% and 58.8%, respectively (95% CI: 43.0%-74.5%). Conclusion: HER2-targeted ADCs showed great survival benefits in GC/GEJC patients as second- and later-line treatments. However, the relatively high incidence of grade≥3 AEs needs to be considered.

Objectives To characterise the epidemiology of atopic eczema in adolescents from Kosovo, an area of very low prevalence of the condition and to know whether the same factors are associated to eczema with or without comorbid wheezing symptoms. Methods The cross-sectional survey Global Asthma Network validated questionnaire was self-completed by adolescents 13-14 years of age in the school setting from the main cities of Kosovo: Ferizaj, Gjakova, Gjilan, Peja, Prishtina and Prizren. Atopic eczema symptoms and diagnosis was put in relation with the environmental questionnaire which included questions on smoking; pet ownership; paracetamol use, truck traffic; siblings; time spent using screens or watching television; and exercise. Additionally, height and weight were measured at school. Results The prevalence of eczema symptoms ranged from 2.2% in Ferizaj to 5.5% in Gjakova. Severe symptoms were <1% in all cities. Eczema ever ranged from 3.0% in Ferizaj to 6.4% in Prizren. Factors significantly associated to the prevalence of current eczema symptoms in the metanalysis were male sex (pooled aOR 0.50; 95%CI 0.37-0.66); exercise (pooled aOR 2.79; 95%CI 1.89-4.10); and paracetamol intake (pooled 1.86; 95%CI 1.32-2.64). The corresponding figures for eczema ever were: 0.68 95%CI 0.44-1.06; 2.07 95%CI 1.48-2.90; and 1.19 95%CI 0.88-1.60. The associations tended to be higher in the subpopulation with eczema and wheeze comorbidity. Conclusions The prevalence of atopic eczema is very low in Kosovo and is associated to female sex, exercise, and paracetamol intake. Those associations are higher when eczema and wheezing are comorbid conditions.

Objective:Population pharmacokinetics analysis explored the pharmacokinetics of anlotinib in children with soft tissue sarcomas (STS) and identified the optimal dose for children across various age brackets. Method:From 2021 to 2023, a single dose of anlotinib (4.62 mg/m2) was orally administered in 16 children with advanced STS in 8 days. Anlotinib plasma concentration was evaluated by LC-MS/MS. Pharmacokinetic models were developed using nonlinear mixed-effects modelling. The effect of predefined covariates on pharmacokinetic parameters was assessed. Results:Totally 128 samples from 16 children (aged 5-14) were collected for pop-PK analysis. The two-compartment model was most consistent with the data of oral anlotinib in pediatrics with advanced STS, and the relevant parameters were: Ka (h-1) 0.419; Vc/F (L) 760; Q (L∙h-1) 21.2; Vp/F (L) 547. Covariate screening showed that the clearance of anlotinib gradually increased with age in a sigmoidal relationship, the maximum CL/F was 15.7L∙h-1, and age of median clearance (Age50) was 6.84 years; the Vc/F increased linearly with BSA. Dose of 8 mg anlotinib for children aged 5-7, and 10 mg or 12 mg for children aged 8-10 would be expected to lead to a similar exposure of anlotinib compared with an adult patient receiving 12 mg. Conclusion:The population pharmacokinetics of orally administrated anlotinib were evaluated in pediatric advanced STS patients. BSA and age were significant physiologic factors on PK. A simulation of 8 mg anlotinib in children aged 5-7, 10 mg or 12 mg in 8-10 and 12 mg for children over 11 would get similar exposure of adults receiving 12mg.

Background IL-4 and IL-13, pivotal components of type 2 immunity, have been implicated in inducing trained immunity in myeloid cells. However, the precise inflammatory response of monocytes in individuals with type 2 immune disorders remains uncertain. Objective To investigate the nuanced inflammatory response of monocytes in patients with type 2 immune disorders, specifically atopic dermatitis and urticaria, upon exposure to LPS stimulation. Methods We collected peripheral blood mononuclear cells (PBMCs) from patients diagnosed with atopic dermatitis and urticaria. Monocytes were isolated from PBMCs and subjected to targeted LPS stimulation. RNA extraction followed by RNA-seq and ATAC-seq analyses were conducted to elucidate molecular responses. Results Monocytes from patients with type 2 immune disorders exhibited an augmented inflammatory response upon LPS stimulation. RNA-seq analysis demonstrated significant upregulation of inflammatory cytokines, including IL-6, IL-12, and IL-1b. Moreover, there was a conspicuous enrichment of signaling pathways associated with inflammation. ATAC sequencing revealed heightened chromatin accessibility in patient monocytes, particularly in the promoter regions of inflammatory genes. Conclusion Our findings unveil an intensified pro-inflammatory response in monocytes following LPS stimulation in type 2 immune disorders such as atopic dermatitis and urticaria. The comprehensive RNA and chromatin analyses underscore the pivotal role of type 2 immune responses in shaping the epigenetically defined inflammatory phenotypes of peripheral monocytes.

Super duplex stainless steels (SDSS) have an ideally dual-phase microstructure composed of austenite and ferrite. However, due to variations in cooling rate, secondary phases (SP), such as sigma, may arise, jeopardising the fatigue performance. This work investigates the correlation between macroscale fatigue behaviour and the multiphase microstructure in SDSS. Fatigue tests were conducted using miniature specimens representative of three regions of a cast C-Ring with different cooling rates, meaning distinct SP amounts. A numerical model was proposed to assess the fatigue test parameters. SEM and EBSD techniques were used to quantify the SP and analyse the fatigue-crack propagation, complemented by hardness and tensile test results. Regression models were developed to correlate the percentage of SP, the fatigue resistance and the number of cycles to failure, and to predict the microstructural stress concentrations from the amount of SP. In addition, a methodology is proposed for predicting S-N curves as a function of the amount of SP. The results show a high correspondence in the increase of the percentage of SP with the decrease in fatigue life. The fatigue strength, for a given number of cycles to failure, tends to decrease linearly with increasing the SP. The microstructural stress concentration factor tends to increase with increasing SP. The research described in this manuscript contributes to further knowledge on predicting the behaviour of SDSS under different cyclic conditions, which can be valuable for optimizing design and performance in various applications.

Skin-like electronics research aiming to mimic even surpass human-like specific tactile cognition by operating perception-to-cognition-to-feedback of stimulus to build intelligent cognition systems for certain imperceptible or inappreciable signals was so attractive. Herein, we constructed an all-in-one wearable device to address the sensitivity and power supply simultaneously by integrating multistage micro-structured ionic skin (MM i-skin) and piezoelectric & thermoelectric dual-mode self-power staffs. The MM i-skin with multi-stage “interlocked” configurations achieved precise recognition of subtle signals, where the sensitivity reached up to 77.4 kPa-1, as well as response time of 46 ms, cyclic stability (over 1500 cycles), and a pressure detection limit (over 50 kPa). Furthermore, we developed a piezo/thermo-electricity dual-mode nanogenerator, denoted as P-iskin / T-iskin, for self-powering the pressure sensor. This self-powered multifunctional ionic skin enables real-time monitoring of weak body signals, rehab guidance, and robotic motion recognition, demonstrating potential for internet of things (IoT) applications involving the artificial intelligence-motivated sapiential healthcare internet (SHI) and widely distributed human-machine interaction (HMI).

Background: Adverse effects resulting from Capecitabine-based chemotherapy pose a significant concern in clinical practice. While the pharmacokinetics (PK) approach has been commonly used to investigate the toxicity of Capecitabine, its predictive ability remains limited. This study aims to assess whether pre-chemotherapy endogenous plasma metabolome can offer improved predictive values for Capecitabine chemotherapy-related adverse events (CRAEs). Methods: Plasma samples were collected from colorectal cancer patients at different time points: 0 hours (before), and 1, 2.5, 4 hours after oral Capecitabine administration, to assess individual variations in exposure levels of Capecitabine and its metabolites. Additionally, the endogenous metabolome profile was analyzed using UHPLC-MS/MS and UHPLC/Q-TOF-MS. Results: Capecitabine and its metabolites can predict two CRAEs, with 5-FU, 5’-DFCR, and FUH2 exposures being associated with diarrhea and thrombocytopenia, respectively. In contrast, identified plasma engougenous biomarker metabolites can predict all seven observed CRAEs. These CRAE-related endogenous plasma metabolites are involved in various physiological functions, including cell proliferation, maintenance, and inflammation. Pre-chemotherapy endogenous plasma metabolites established superior predictive performance for CRAEs (AUROC values ranging from 0.834 to 0.984) compared to conventional drug exposure (AUROC values ranging from 0.737 to 0.773). Additionally, the endogenous plasma metabolome demonstrated a strong correlation with Capecitabine and its metabolite exposures. Conclusions: Our study demonstrates that pre-chemotherapy endogenous plasma metabolome serve as superior biomarkers for predicting Capecitabine-related adverse effects, surpassing the value of exposures to Capecitabine and its metabolites. This finding holds significant potential in guiding personalized medicine approaches.

Dry fractionation is an environmentally friendly and cost-effective method to separate protein fractions. While most research has focused on the structure and functional properties of pea protein isolates (PPI), the information on dry fractionated pea protein (DFPP) is limited. This study compared DFPP (protein content (PC)-50.7%, insoluble fiber-17%), to PPI (PC-80.1%, insoluble fiber-8.33%), in terms of the protein structure, solubility, and heat-induced gelation. SDS-Page, size-exclusive chromatographic, and Fourier-transform infrared spectrophotometer analysis indicated that DFPP contained the major protein components in pea, and their native structures were well maintained. Whereas for PPI, some proteins were lost during wet extraction, and partial protein unfolding and aggregation were observed. At neutral pH, DFPP showed significantly higher solubility (44.64±0.55%) than PPI (12.09±1.42%). Interestingly, DFPP showed good gelling capacity as reflected by lower gelling concentration and higher gel mechanical strength and elasticity compared to those made from PPI. The DFPP gels were twice higher in mechanical strength (7.71±0.21kPa) than that prepared from PPI at pH 7. Strong gels were also obtained for DFPP at pH 5. The gel morphology revealed phase separation between protein and polysaccharides by heating, with stick-shaped fiber (10-25μm) dispersed in the continuous protein networks. Eventually, the polysaccharides including fiber and starch helped strengthen the gel network by acting as fillers. This knowledge will help to expand the applications of DFPP as a gelling ingredient in food formulations, but also allow industry to benefit from the dietary fiber co-exist in the protein to develop healthier food products using a holistic approach.

Background:With the mounting number of intrauterine operations, the incidence of intrauterine adhesion continues to rise. However, the specific pathogenesis of intrauterine adhesion has not been clear and there is no fundamental treatment so far. Objective:IUA is an endometrial fibrosis illness that gravely jeopardizes the reproductive health of women of reproductive age. We elucidated the pathogenesis of IUA at the protein level to provide a more effective research basis for diagnosis and treatment. Methods: This study collected 15 endometrial tissue samples and divided them into three comparison groups (Adhesion/Control group, Endometrium/Adhesion group, and Endometrium/Control group). Label-free quantitative proteomics was used to identify differentially expressed proteins and performed a comprehensive bioinformatics analysis. The research was followed by a quantitative analysis of selected target proteins using a parallel reaction monitoring method. Results: 1328, 290, and 1335 differential proteins were found in each comparison group. Many features of the differential proteins were also identified by GO enrichment analysis and KEGG pathways analysis. Parallel reaction monitoring analysis was performed to quantify 14 target proteins (PGM5, CAMK2G, ABHD6, MYLK, SYNPO2, LTF, PTPN11, TGF-β1, SLC4A1, PROS1, IRAG1, ADIPOQ, HLA-DRB3, COQ6). Among them, TGF-β1 was previously reported as a significant protein in intrauterine adhesion, while other proteins were newly discovered. Conclusions: These proteins are anticipated to develop as biomarkers for intrauterine adhesion and have a specific role in diagnosis and therapy.

Breast granular cell tumor: A report of two cases and review of literatureAssim Saad Eddin, MD1; Umar Ramzan, BS2; Su Kim Hsieh, MD, PhD1; Fabiana, Policeni, MD1University of Iowa Hospitals and Clinics, Department of Radiology, Iowa city, Iowa, United StatesNorthwestern Feinberg school of medicine, Chicago, Illinois, United StatesCorresponding author:Assim Saad Eddin, MD738 mission point road, 52245, Iowa city, Iowa, United StatesResearch ScholarDepartment of RadiologyUniversity of Iowa Hospitals and ClinicsKeywords: Radiology and ImagingWord count: 2107Key clinical message:Granular cell tumor (GCT) is a rare breast neoplasm. There are only a few reports of breast granular cell tumors in the literature. Here in, we present two cases of female patients diagnosed with this tumor and perform a review of literature on the prevalence, diagnosis, histology, treatment, and prognosis\(.\)

The study focused on the impact and action mechanism of different organic selenium products on the bioactivity of two strains of Lactobacillus paracasei. The growth, antioxidant activity, extracellular polysaccharide secretion, quorum sensing and biofilm formation of the strains before and after the addition of organic selenium crude products and three organic selenium standard were evaluated. The results showed that the addition of crude organic selenium had a promoting effect on the growth, antioxidant activity, extracellular polysaccharide, biofilm formation, and AI-2 activity of the strain. The organic selenium standards had a regulatory effect (positive and negative) on biofilm formation and the activity of the QS signal molecule AI-2 in the strain. L-selenocysteine had the strongest regulatory effect, with maximum GIM1.80 biofilm formation when it reached a critical concentration of 0.4 μg/ml; L-selenomethionine resulted in the highest activity of the signal molecule AI-2 of GDMCC1.155, when it reached a critical concentration of 0.4 μg/ml. The results of scanning electron microscopy demonstrated that organic selenium effectively improved the morphology and structure of the bacterial cells of the two strains. Molecular docking revealed that the mechanism by which organic selenium regulates quorum sensing in Lactobacillus was achieved by binding two crucial receptor proteins (histidine protein kinase HKP and periplasmic binding protein LuxP) from specific sites. It provides a new alternative (organic selenium) for regulating the viability and beneficial activity of Lactobacillus paracasei. Significance statement: The study found that organic selenium can both positively and negatively regulate quorum sensing and biofilm formation of Lactobacillus, and the critical concentrations have also been obtained. Meanwhile, it was found that organic selenium can promote the growth, antioxidant activity, and biofilm formation of Lactobacillus by activating quorum sensing, so as to better exert its probiotic properties. Furthermore, the regulating mechanism of organic selenium on the activity of Lactobacillus was revealed. This study provides a new alternative for regulating and stimulating the beneficial activity of probiotics.

It is almost universally thought that modulation of immune function by tryptophan (Trp) metabolites involves activation of the aryl hydrocarbon receptor (AhR) mainly by kynurenine (Kyn), based on enhanced expression of cytochrome P-450 enzymes and their increased activities in cell systems and in vivo. However, DiNatale et al. (Toxicol. Sci. 2010; 115: 89-97) reported the failure of Kyn at 10 M to activate the AhR, whereas a similar concentration of kynurenic acid (KA) was effective. The recent study by Solvay et al (J Immunother Cancer 2023; 11: e006728) called into question the direct link between Kyn and the AhR and demonstrated down regulation of the AhR by Trp. In the present study, we have performed for the first time molecular docking in silico to the human AhR of the above and a range of other Trp metabolites produced in the various degradative pathways and by gut microbiota. We demonstrate that, of 29 Trp metabolites, only Kyn and 3-hydroxykynurenine fail to dock to the AhR and propose that AhR activation by Kyn is an indirect effect mediated by KA. The strongest docking is observed with FICZ (6-Formylindolo[3,2-b]carbazole), cinnabarinic acid, 5-hydroxytryptophan, N-acetyl serotonin and indol-3-yllactic acid. We propose that Trp, which docks strongly to the AhR is an AhR antagonist. Differences in AhR activation by Trp metabolites in cell systems and in vivo may be determined by the prevailing physiological conditions. The strong docking of 5-hydroxyindoles to the AhR may underpin the effects of serotonin pathway metabolites on biological processes.

Due to the widespread use of engineered nanomaterials (ENMs) for soil remediation and nano-enabled sustainable agriculture, there is a growing concern regarding the behavior and fate of ENMs released into soil systems in the presence of natural root exudates (REs). Herein, we investigate the influence of REs on the fate and ecological effect of ENMs from a comprehensive perspective. We summarize the key roles reported in the literature for REs in physical changes (e.g., adsorption, dispersion/aggregation), chemical changes (e.g., oxidation/redox reactions, and dissolution), and biotransformation of ENMs, which will further determine the ecological risk of ENMs in natural soil systems. Moreover, this review highlights the potential adverse effects of ENMs on different soil organisms (e.g., bacteria, plants, and eisenia foetida) in the presence of REs. The remaining unclear mechanisms (e.g., oxidative stress and DNA damage) of ENMs toxicity at the cellular level influenced by REs are reviewed and presented. Finally, the review concludes by addressing the current knowledge gaps and challenges in this field.

Evaluation of flow dynamics in dSINE (distal Stent graft-induced New Entry) using 4D flow MRIKenji Sakakibara1), Hiroyuki Nakajima1), Yudai Hagihara1), Chie Nakamura1), Daichi Shikata1), Yuki Takesue1), Satoru Shiraiwa1),Yoshihiro Honda1),Shigeaki Kaga1)Masahiro Hamasaki2), Hisashi Johno2)1) Department of Surgery (II), Faculty of Medicine, University of Yamanashi2) Department of Radiology, Faculty of Medicine, University of Yamanashi

This study evaluated erosion rates and sediment production in streams, and factors potentially influencing them throughout the Anacostia, Patuxent, and Potomac (non-Anacostia) River watersheds within Prince George’s County, Maryland, US. As part of the County’s watershed-scale biological monitoring program, from approx. 1999-2008, permanent monuments were established to allow measurement of stream channel cross-sectional (XS) area. The intent of this study was to characterize the intensity and spatial distribution of fluvial geomorphic instability across the county and use the results to target and plan stormwater management and stream restoration actions. For this study, 78 stream locations were re-surveyed in 2020, representing a time lapse of from 12-21 years. Data collected included XS dimensions, modified Wolman 100-particle pebble counts, and reach-specific soil bulk density. Land use/land cover data were compiled from the National Land Cover Dataset (NLCD), precipitation from the National Weather Service Center for Environmental Information (NCEI), and soils from the Natural Resources Conservation Service Web Soil Survey (NRCS/WSS). We calculated percent change in XS area, rates of erosion, sediment yield, and assigned geomorphic classifications, and interpreted them in the context of spatial positions relative to changes in land cover characteristics. Sediment yields among the 78 reaches exhibited a combination of those undergoing degradation/erosion (67.9%), aggradation/deposition (25.6%), and the remaining 6.4% with essentially no change over the period of record. Of the top 20 most geomorphically active reaches surveyed in the County, 12 are in the Anacostia River basin, with the other scattered among the Patuxent River and Potomac River basins.

Roegneria yenchiana sp. nov. (Triticeae) is described as a new species collected from Shangri-la of Yunnan Province in China based on morphological, cytological, and molecular data. It is morphologically characterized by one spikelet per node, rectangular glums, awns flanked by two short mucros in lemmas, which is distinguished from other species of Roegneria. The genomic in situ hybridization results indicate that R. yenchiana is an allotetraploid, and its genomic constitution is StY. Phylogenetic analyses based on multiple loci suggested that R. yenchiana is closely related to Pseudoroegneria and Roegneria, and the Pseudoroegneria served as the maternal donors during its polyploid speciation.

This study examines the role of merit in the concept of corporate social responsibility. The text outlines the mechanisms of support for those who are perceived by society as undeserving of the aid in question. Its aim is to analyze the context of the decision-making process in companies on the one hand and nonprofit organizations on the other. A qualitative approach has been chosen to achieve the goal, which reveals the ways in which the partakers think. The study examines the legitimacy of the choice of the target group, the importance of the community in the local context, and the strategy of communication support. The results of the study identify four different mechanisms of support from companies, including support through a foundation, the company's own foundation, the employee volunteer association, and a consortium of the company and nonprofit organization. At the same time, the research has concluded that the various methods of support intersect and together create a comprehensive mechanism with a number of stakeholders, which can only work under the conditions of mutual consensus.

Microfiltration is an essential step during biopharmaceutical manufacturing. However, unexpected flux decay can occur. Although the flux decay profile and initial flux are important factors determining microfiltration filterability, predicting them accurately is challenging since the root cause of unexpected flux decay remains elusive. In this study, the methodology for developing a prediction model of flux decay profiles was established. First, the filtration profiles of different monodisperse polystyrene latex and silica beads of various sizes were evaluated. These results revealed that the size and surface electrostatic properties of the beads affect the flux decay profile. Taking the size and surface electrostatic properties of protein aggregates into account, we constructed a predictive model using model bead filtration profiles. We showed that this methodology was applicable to two different microfiltration filters to predict the flux decay profile of therapeutic proteins. Since this prediction model is based on normalized flux, the initial flux must be predicted. We therefore successfully developed a method to predict initial flux based on the Hagen-Poiseuille equation using sample viscosity values for both filters. These prediction models can be used for effective microfiltration scale-up assessment and can be applied during early stage of process development.

The blood protein von Willebrand factor (VWF) is a large multimeric protein that, when activated, binds to blood platelets tethering them to the site of vascular injury initiating blood coagulation. This process is critical for the normal haemostatic response, but especially under inflammatory conditions it is thought to be a major player in pathological thrombus formation. For this reason, VWF has been the target for the development of anti-thrombotic therapeutics. However, it is challenging to prevent pathological thrombus formation while still allowing normal physiological blood coagulation as currently available anti-thrombotic therapeutics are known to cause unwanted bleeding in particular intracranial haemorrhage. This work explores the possibility of inhibiting VWF selectively under the inflammatory conditions present during pathological thrombus formation. In particular, the A2 domain of VWF is known to inhibit the neighboring A1 domain from binding to the platelet surface receptor GpIb α and this auto-inhibitory mechanism has been shown to be removed by oxidizing agents released during inflammation. Hence, finding drug molecules that bind at the interface between A1 and A2 only under oxidizing conditions could restore such auto-inhibitory mechanism. Here, by using a combination of computational docking, molecular dynamics simulations and free energy perturbation calculations, a ligand from the ZINC15 database was identified that binds at the A1A2 interface with the interaction being stronger under oxidizing conditions. The results provide a framework for the discovery of drug molecules that bind to a protein selectively in inflammatory conditions.

Histidine kinases (HKs) are a central part of bacterial environmental-sensing two component systems, providing their hosts with the ability to respond to a wide range of physical and chemical signals, being a paradigmatic example of protein diversity and molecular evolution. HK are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp) and a catalytic domain. They work as homodimers, and the existence of two different autophosphorylation mechanisms (cis and trans), has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs, is still unavailable, nor is there any evolutionary analysis on the subject. In this work we show that AlphaFold can accurately determine whether a HK dimerizes in a cis or trans structure. We then use it to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch, providing these systems with a way to diverge rapidly and adapt to new stimuli.

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.