Treponema pallidum subsp. pallidum , the causative agent of syphilis, remains difficult to study due to long–standing limitations in in vitro cultivation. Although a rabbit epithelial cell co–culture system (Sf1Ep) enabled major advances in recent years, the lack of human cell–based models restricts clinical relevance and mechanistic insight into host–pathogen interactions. Here, we sought to establish human epithelial cell lines capable of supporting T. pallidum growth in vitro . Six human epithelial or epithelial-like cell lines from diverse tissue origins were evaluated under microaerophilic conditions using the standard T. pallidum cultivation medium. Among these, CAL–39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviours, and long–term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility of T. pallidum were quantified over extended culture periods. Using live–cell imaging, we define for the first time two distinct T. pallidum host-cell interaction behaviors — surface–associated crawling and stable single–polar attachment. Both behaviours were observed across all tested host cell lines and persisted over time only in cell lines permissive for sustained growth. Together, our findings establish clinically relevant human epithelial co–culture models for T. pallidum , provide new insights into host–cell–dependent growth and motility, and create a platform for future mechanistic studies of syphilis pathogenesis and vaccine target discovery. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council (BBSRC), BB/010589 Bill & Melinda Gates Foundation, INV-051483 Wellcome funding, 220540/Z/20/A

Phage therapy offers an alternative to antibiotics for treating multidrug-resistant infections. Plasmid-dependent phages (PDPs) are promising therapeutics as they can kill targeted pathogens and prevent the spread of plasmid-encoded antibiotic resistance genes. However, the evolutionary trajectories of multidrug-resistance (MDR) plasmids under the selective pressure of PDPs remain poorly understood, particularly in eco-evolutionary contexts that remain permissive to plasmid conjugation. We experimentally evolved populations of Escherichia coli carrying the MDR plasmid RP4 in the presence of the plasmid-dependent phage PRD1 under conditions where the benefits of conjugation were either strong or weak. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of phage-resistant plasmids lacking conjugative ability. Increasing ecological opportunities for conjugation enhanced plasmid suppression and delayed the evolution of phage-resistant plasmids. PRD1 resistance was associated with plasmid loss and reduced conjugative ability, though this trade-off was complex because resistance mutations caused heterogeneous effects on pilus production and conjugation. Mutations and insertion sequence (IS)-mediated inactivation in conjugation genes generated a spectrum of resistance phenotypes, from partial (trbB, trbL) to complete (virB4/trbE) resistance. Bioinformatic analysis of publicly available IncP plasmids revealed frequent truncations of the VirB4/TrbE protein, suggesting that plasmid-dependent phages may represent an important selective pressure shaping plasmid evolution in natural populations. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that plasmids cannot easily circumvent. Targeting multidrug-resistance plasmids with PDPs is likely to drive loss of conjugation, limiting the transfer of antibiotic resistance genes in microbial communities.

Treponema pallidum subsp. pallidum, the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances, the absence of human cell-based models limits clinical relevance and mechanistic understanding of host-pathogen interactions. Here, we established a co-culture system using human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six epithelial or epithelial-like cell lines from diverse tissues were evaluated under microaerophilic conditions using standard cultivation medium. CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviours, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility were quantified over extended culture. Using live-cell imaging, we defined two distinct host-cell interaction behaviours: surface-associated crawling and stable single-polar attachment. Both behaviours were observed across all tested cell lines but persisted only in those permissive for sustained growth. Together, our findings establish clinically relevant human epithelial co-culture models for T. pallidum, provide new insights into host-cell-dependent growth and motility, and create a platform for mechanistic studies of syphilis pathogenesis and vaccine target discovery.

Polyamines are a group of organic compounds that can be found in a variety of foods such as meat, fish, vegetables, and milk products including infant formula. Existing methods developed for the quantification of polyamines in infant formulas do not assess the matrix effect and therefore are potentially under- or over- quantifying the polyamine content. Infant formula is a specialized food group where accurate quantification of polyamines is desired. In the current study, a detailed analytical method for the quantification of polyamines in an infant formula matrix was developed using reverse-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV). The matrix effect was also evaluated during method development. The new method was validated in terms of trueness, precision, linearity, LOD/LOQ and uncertainty. LODs ranged from 80 to 200 ppb and LOQs ranged from 240 to 600 ppb. Correlation coefficients ranged from R2= 0.99-1.0. A fit for purpose, widely accessible analytical method for the absolute quantification of polyamines in an infant formula matrix now exists.

Phage therapy is a promising alternative to antibiotics to treat multidrug resistant infections. Plasmid dependent phages (PDPs) are particularly attractive as therapeutics because they can both kill targeted pathogen cells, whilst also potentially preventing the further spread of antibiotic resistance genes encoded by plasmids. However, we lack experimental studies of the ecological and evolutionary response of multidrug resistance plasmids against plasmid dependent phage treatment under ecologically relevant scenarios allowing plasmid conjugation. We experimentally evolved populations of E. coli carrying the multidrug resistance RP4 plasmid with the PRD1 PDP under conditions where conjugation was associated with either strong or weak benefits. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of PRD1 resistant plasmids that lacked conjugative ability. Increasing the ecological opportunity for conjugation enhanced plasmid suppression and delayed the evolution of PRD1 resistant plasmids. PRD1 resistance was associated with reduced conjugative ability, but this trade-off was complex due to the heterogeneous impacts of resistance mutations on pilus production and conjugative ability. Mutations and IS element insertions in conjugation genes caused a wide range of PRD1 resistance phenotypes, ranging from complete resistance ( virB4 ) to partial resistance ( trbB , trbL ). Bioinformatic analysis of publicly available IncP plasmid sequences showed that truncated variants of VirB4 protein are common in natural populations, suggesting that plasmid-dependent phages are an important selective pressure in microbial communities. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that cannot be easily circumvented by plasmids. Targeting multidrug resistance plasmids with PDPs is likely to drive loss of conjugation limiting the transfer of antibiotic resistance genes in bacterial communities. ### Competing Interest Statement The authors have declared no competing interest. UKRI, MR/W031361/1 UKRI Frontiers Grant, EP/Y031067/1

Immunotherapy has transformed cancer treatment but benefits only some patients, and predictive biomarkers are lacking. One correlate of response is the reinvigoration of a subset of CD8 T cells that have an exhausted phenotype and impaired functionality. To develop effective therapies, reproducible models are required to identify candidate target genes that enable reversal of T cell exhaustion. Here, we describe an in vitro model by chronically stimulating T cells with their cognate antigen, followed by temporal phenotypic characterization. This model recapitulates many critical hallmarks of exhaustion, including expression of canonical surface markers, impaired proliferation, reduced cytokine production, decreased cytotoxic granule release, and metabolic alterations. Two in vivo models validate these results and establish a gene signature shared by in vitro and in vivo exhausted states. Critically, this signature is observed in tumor infiltrating T cells from multiple human tumor types, validating the translational potential of this model for discovering therapies.

Whey proteins provide nutritional and functional properties in many dairy products, and their denaturation induced by heat has drawn attention in dairy research and industry. Denatured whey proteins have functional properties that are beneficial for certain applications, such as yogurt, while uncontrolled denatured protein aggregation can impair certain processes, such as rennet coagulation. The possible effect of genetic polymorphism of (3-lg and kappa-CN on heat-induced denaturation of whey proteins in milk and in rennet whey was investigated in this study. No effect of kappa-CN genotype on the denaturation of (3-lg and alpha-lac was detected, while the A variant of (3-lg was more heat-stable than the B variant. More denatured (3-lg B was shown to interact with casein micelles to form insoluble aggregates compared to the A variant. These results may provide guidance for selection of specific milk genotypes for applications where whey protein denaturation needs to be minimised or else preferred, such as foaming and emulsification.

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage, and visualised the effects of structural changes in the polar plugs, leading to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing ‘hatch-ready’ L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes.

Milk fat globule membrane (MFGM) proteins are receiving increased attention due to their reported benefits for human health, particularly in infant populations. Challenges exist in MFGM protein quantification due to their low quantities, complex chemistry, interactions with other matrix components, and the high matrix complexity. In this study, a subset of four MFGM proteins were selected as relevant targets for identification and quantification in an infant formula (IF) matrix: butyrophilin, mucin 1, xanthine dehydrogenase/oxidase, and perilipin 2. An analytical protocol for absolute quantification of these four MFGM proteins in IF was successfully developed and validated. Additionally, the feasibility to apply the method in selected MFGM ingredients was also demonstrated. The method demonstrated good performance; high accuracy and robustness, low LOQ and uncertainty, while controlling the matrix effect. This study represents a report of an analytical method to quantify selected MFGM proteins in IF.

This study examined how the genetic variants A1 and A2 of beta-casein (beta-CN) affect proteolytic pathways in bovine milk, specifically examining how the amino acid substitution of histidine (A1) with proline (A2) influences the hydrolysis of milk proteins by plasmin. Raw milk samples from seven cows were collected on three occasions, and their proteolysis was analysed using urea-PAGE and UPLC-UV. Peptides released by proteolysis were identified using LC-MS. Milk with the A2A2 genotype exhibited higher plasmin activity, and caseins were more susceptible to hydrolysis by plasmin than those in A1A1 milk. Peptides from beta-CN His67-Asn68 cleavage were present in A1A1 milk, but no peptides were linked to cleavage sites 66-67 or 67-68 in A2A2 milk. This indicates that a single amino acid difference can significantly influence proteolytic pathways, which may affect dairy processes and product quality.

As the non-protein nitrogen (NPN) fraction of milk, and its components, do not directly contribute to protein synthesis, historically, it was considered to have little biological, nutritional, or economical value. Recently, there has been an increased emphasis on NPN components and their association with health benefits. While the NPN fraction has previously been perceived neutrally, or indeed sometimes negatively, recent studies suggest that the fraction is undervalued or misunderstood, particularly regarding infant growth and development, highlighting the need for a comprehensive understanding of the NPN components in infant formula (IF) and the dairy ingredients most used in its formulation. This study focuses on the identification and quantification of NPN components in selected dairy ingredients and emphasizes the importance of considering NPN during the formulation of IF. Some findings to note are that whole milk powder was a significant source of uric acid, while alpha-lactalbumin enriched whey had the highest concentration of sialic acid. The results provide valuable insights for the scientific community to understand which dairy ingredients represent good innate sources of NPN components in IF, such as nucleotides, peptides, choline and carnitine, which are routinely supplemented in IFs and are associated with various health benefits, including immune support, gut health, and growth and development. The knowledge generated in this study will also allow for informed decision-making for ingredient selection, which will enable the development of IFs with NPN profiles closer to human milk. Additional research opportunities in the NPN area have also been identified.

Lactoferrin (LF) and osteopontin (OPN) form heteroprotein complex coacervates (HPCCs) within limited conditions of pH, ionic strength, total protein concentration (TPC), and protein stoichiometry. In this study, it was determined using dynamic oscillatory rheology, that the coacervates exhibit frequency-dependent, viscoelastic behaviour whereby the storage modulus (G ') was less than the loss modulus (G '') for all frequencies studied (<= 100 rad/s). By reducing the TPC and/or ionic strength of the system, an increase in the protein concentration and strength of the coacervate was achieved. The G ' and G '' of the coacervates were largely independent of temperature <50 degrees C, but above this temperature, significant changes occurred due to protein denaturation. Using microscopic techniques, it was also determined that both proteins co-localise throughout the coacervate phase in a highly ordered, structural network of protein and water. The resulting freeze-dried coacervate powder particles had a honeycomb-like appearance due to an intricate network of porous channels formed during the drying process. This physicochemical characterisation of LF-OPN HPCCs has increased scientific understanding of their rheological and microstructural properties which can now be understood, predicted, and controlled for subsequent biochemical analysis.

BACKGROUND:Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS:Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS:Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

Milk characteristics and suitability for production of dairy products like cheese may be influenced by protein polymorphism; the possible influence of β-casein (β-CN) polymorphism on cheese manufacturing and cheese characteristics was examined using milk with three β-CN genotypes, A1A1, A1A2 and A2A2. The composition and rennet coagulation properties of raw and standardised cheese milk, cheese yield and their composition were examined, cheese meltability, texture and proteolysis were analysed at different ripening times. Milk with β-CN variant A2A2 was found to have poorer rennet coagulation properties following standardisation and pasteurisation. Cheese made from A1A1 milk had lower protein content, while that from A2A2 milk had lower fat content compared with the other two genotypes. There was no significant impact of genotype on proteolysis during cheese ripening. However, β-CN genotype was found to impact cheese texture; after 180-d ripening, the A1A1 cheese was the softest, and A2A2 cheese was the most fracturable.

Sources of milk fat globule membrane (MFGM) are desirable to include in infant milk formula (IMF) to mimic the composition and functionality of human milk MFGM. MFGM in its natural form consists of a trilayer structure containing lipids (e.g., cholesterol, phospholipids, gangliosides, ceramides), proteins (e.g., butyrophilin, xanthine oxidase, mucin-1, adipophilin) and glycans (e.g., sialic acid). Components of MFGM have been associated with various biological benefit areas including intestinal, neurocognitive, and immune health. There are many aspects to consider when supplementing IMF with MFGM ingredients, of which the major ones are highlighted and critiqued in this review from an industrial research perspective. Features include compositional unknowns, discussion on how best to incorporate MFGM to IMF, analytical method needs, biological function unknowns, and considerations on how best to communicate MFGM in different contexts. It is hoped that by identifying the key scientific gaps outstanding in this subject area, collective efforts can proceed to ensure the potential impact of MFGM on infant health is realized.

Lactoferrin (LF) and osteopontin (OPN) are bioactive milk proteins which can form heteroprotein complexes and complex coacervates. This research studied the effect of LF-OPN complexation and complex coacervation on the simulated infant gastrointestinal digestion of LF with subsequent examination of gut and bone health bioactivities in preclinical models. In an infant digestion model, the proteolytic profile of LF was unaltered by the pre-association of LF and OPN. Gastric proteolysis of LF was increased when the model gastric pH was reduced from 5.3 to 4.0, but less so when complexed with OPN. In a model of intestinal inflammation, undigested (79% inhibition) and gastric digestates (26% inhibition) of LF, but not gastrointestinal digestates, inhibited lipopolysaccharide (LPS)-induced NF-kappa B activation in intestinal epithelial cells. LF-OPN complexation sustained the inhibitory effect (21-43% of the undigested effect, depending on the type of complex) of LF after gastrointestinal digestion, suggesting that the peptides produced were different. In a neonatal rodent model used to study bone development, coacervating LF and OPN improved bone structures with a significant increase of trabecular proportion (BV/TV increase by 21.7%). This resulted in an 11.3% increase in stiffness of bones. Feeding the LF and OPN proteins in coacervate format also increased the levels of OPN, P1NP and M-CSF in blood, signifying a more pronounced impact on bone development. This research demonstrated that LF-OPN complexation and complex coacervation can delay simulated infant gastrointestinal digestion of LF and protect or improve the bioactivity of the proteins.

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.

ABSTRACTThe recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss‐of‐function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red‐shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy‐ and cell sorting–based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

Lactoferrin is of interest for infant nutrition due to its roles in supporting gastrointestinal development. When supplemented orally, digestive exposure may reduce the availability and biofunctionality of the protein and its derived peptides. The aim of this study was to evaluate the proteolysis of bovine lactoferrin and β-casein coacervates following infant in vitro digestion. Coacervation of β-casein with lactoferrin showed a protective function against peptic proteolysis, increasing the levels of intact β-casein and lactoferrin during gastric digestion, while the uncomplexed β-casein was readily proteolyzed. The coacervate was shown to protect the C-terminal of β-casein against gastric proteolysis. This resulted in altered peptide profiles in the gastric and intestinal phases, requiring additional research to ascertain potential implications for biological activity. This study demonstrates the impact of protein–protein interactions on proteolysis during digestion, which may delay proteolysis and possibly protect the bioactive properties of the proteins.

Cytochromes P450 can metabolize endogenous fatty acids, such as arachidonic acid, to bioactive lipids such as epoxyeicosatrienoic acids (EETs) that have beneficial effects. EETs protect hearts against ischemic damage, heart failure or fibrosis; however, their effects are limited by hydrolysis to less active dihydroxy oxylipins by soluble epoxide hydrolase (sEH), encoded by the epoxide hydrolase 2 gene (EPHX2, EC 3.3.2.10). Pharmacological inhibition or genetic disruption of sEH/EPHX2 have been widely studied for their impact on cardiovascular diseases. Less well studied is the role of increased EPHX2 expression, which occurs in a substantial human population that carries the EPHX2 K55R polymorphism or after induction by inflammatory stimuli. Herein, we developed a mouse model with cardiomyocyte-selective expression of human EPHX2 (Myh6-EPHX2) that has significantly increased total EPHX2 expression and activity. Myh6-EPHX2 hearts exhibit strong, cardiomyocyte-selective expression of EPHX2. EPHX2 mRNA, protein, and epoxide hydrolysis measurements suggest that Myh6-EPHX2 hearts have 12-fold increase in epoxide hydrolase activity relative to wild type (WT) hearts. This increased activity significantly decreased epoxide:diol ratios in vivo. Isolated, perfused Myh6-EPHX2 hearts were not significantly different from WT hearts in basal parameters of cardiac function; however, compared to WT hearts, Myh6-EPHX2 hearts demonstrated reduced recovery of heart contractile function after ischemia and reperfusion (I/R). This impaired recovery after I/R correlated with reduced activation of PI3K/AKT and GSK3β signaling pathways in Myh6-EPHX2 hearts compared to WT hearts. In summary, the Myh6-EPHX2 mouse line represents a novel model of cardiomyocyte-selective overexpression of EPHX2 that has detrimental effects on cardiac function