Cultivation of phosphate-accumulating biofilm: Study of the effects of acyl-homoserine lactones (AHLs) and cyclic dimeric guanosine monophosphate (c-di-GMP) on the formation of biofilm and the enhancement of phosphate metabolism capacity.

This study investigated the mechanisms of microbial growth and metabolism during biofilm cultivation in the biofilm sequencing batch reactor (BSBR) process for phosphate (P) enrichment. The results showed that the sludge discharge was key to biofilm growth, as it terminated the competition for carbon (C) source between the nascent biofilm and the activated sludge. For the tested reactor, after the sludge discharge on 18 d, P metabolism and C source utilization improved significantly, and the biofilm grew rapidly. The P concentration of the recovery liquid reached up to 157.08 mg/L, which was sufficient for further P recovery via mineralization. Meta-omics methods were used to analyze metabolic pathways and functional genes in microbial growth during biofilm cultivation. It appeared that the sludge discharge activated the key genes of P metabolism and inhibited the key genes of C metabolism, which strengthened the polyphosphate-accumulating metabolism (PAM) as a result. The sludge discharge not only changed the types of polyphosphate-accumulating organisms (PAOs) but also promoted the growth of dominant PAOs. Before the sludge discharge, the necessary metabolic abilities that were spread among different microorganisms gradually concentrated into a small number of PAOs, and after the sludge discharge, they further concentrated into Candidatus_Contendobacter (P3) and Candidatus_Accumulibacter (P17). The messenger molecule C-di-GMP, produced mostly by P3 and P17, facilitated P enrichment by regulating cellular P and C metabolism. The glycogen-accumulating organism (GAO) Candidatus_Competibacter secreted N-Acyl homoserine lactones (AHLs), which stimulated the secretion of protein in extracellular polymeric substances (EPS), thus promoting the adhesion of microorganisms to biofilm and improving P metabolism via EPS-based P adsorption. Under the combined action of the dominant GAOs and PAOs, AHLs and C-di-GMP mediated QS to promote biofilm development and P enrichment. The research provides theoretical support for the cultivation of biofilm and its wider application.

The application of Phase 0 and microtracer approaches in early clinical development: past, present, and future.

Phase 0 microdosing studies were introduced to the drug development community approximately 20 years ago. A microdose is defined as less than 1/100th of the dose calculated based on animal data to yield a pharmacological effect in humans, with a maximum of 100 µg, or 30 nmoles for protein products. In our experience, Phase 0 microdose studies have not been fully embraced by the pharmaceutical industry. This notion is based on the number of Phase 0 studies that we have been involved in. Thus, we conducted at least 17 Phase 0 microdose studies in the Zero's (on average, two per year), but in the years beyond this, it was only 15 studies (1.4 per year); in these latter years, we did conduct a total of 23 studies which employed an intravenous (i.v.) microdose for absolute bioavailability (ABA) assessments (two per year on average), which are the most used and potentially informative type of clinical study using a microdose, albeit they are formally not microdose studies. In the current review, we summarize the past use of and experience with Phase 0 microdose designs in early clinical development, including intravenous 14C microdose ABA studies, and assess what is needed to increase the adoption of useful applications of Phase 0/microdose studies in the near future.

Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis and chronic obstructive pulmonary disease. Prolonged infection allows accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonising upper airway environments. Here, we model this process using an experimental evolution approach. P. aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments - sinus and lungs, under CF and non-CF conditions - selected for loss of twitching motility, increased resistance to multiple antibiotic classes and a hyper-biofilm phenotype. These traits conferred increased airway colonisation potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Decoding the influence of low temperature on biofilm development: The hidden roles of c-di-GMP.

Biofilms are widely used and play important roles in biological processes. Low temperature of wastewater inhibits the development of biofilms derived from wastewater activated sludge. However, the specific mechanism of temperature on biofilm development is still unclear. This study explored the mechanism of temperature on biofilm development and found a feasible method to enhance biofilm development at low temperature. The amount of biofilm development decreased by approximately 66 % and 55 % at 4 °C and 15 °C, respectively, as compared to 28 °C. The cyclic dimeric guanosine monophosphate (c-di-GMP) concentration also decreased at low temperature and was positively correlated with extracellular polymeric substance (EPS) content, formation, and adhesion strength. Microbial community results showed that low temperature inhibited the normal survival of most microorganisms, but promoted the growth of some psychrophile bacteria like Sporosarcina, Caldilineaceae, Gemmataceae, Anaerolineaceae and Acidobacteriota. Further analysis of functional genes demonstrated that the abundance of functional genes related to the synthesis of c-di-GMP (K18968, K18967 and K13590) decreased at low temperature. Subsequently, the addition of exogenous spermidine increased the level of intracellular c-di-GMP and alleviated the inhibition effect of low temperature on biofilm development. Therefore, the possible mechanism of low temperature on biofilm development could be the inhibition of the microorganism activity and reduction of the communication level between cells, which is the closely related to the EPS content, formation, and adhesion strength. The enhancement of c-di-GMP level through the exogenous addition of spermidine provides an alternative strategy to enhance biofilm development at low temperatures. The results of this study enhance the understanding of the influence of temperature on biofilm development and provide possible strategies for enhancing biofilm development at low temperatures.

Microbes identified from monitoring cell manipulations in 5-year life of the Cell Factory G. Gaslini.

Introduction: Quality and safety of a cell product, essential to guarantee the health of patients, depends on many factors including an appropriate environmental monitoring of the manufacturing rooms. Nonetheless, the maintenance of a controlled environment is requested to minimize the risk of contamination. Thus, a timely detection of changes in microbiological trends is important to adopt promptly effective measures against resistant strains that, in turn, may invalidate not only the sanitization procedures but also the safety of the cell product. Methods: We analyzed microbes found in our cell processing clean room over the last 5 years. We used 10.147 plates for air sampler, passive air monitoring and for checking instruments and operators of the production unit. Results: From these plates, 747 colonies were subjected to identification by the MALDI-TOF Vitek® MS system and the large majority of them was gram positive (97.8%) as witnessed by the finding that the most represented genera harvested from the classified areas were Staphylococcus (65%), Micrococcus (13%), Kocuria (8%) and Bacillus (5%). We never detected fungi. Most microbes found in the operators (both from class A and B) were collected from forearms and resulted of the Staphylococcus genus. Conclusions: The observed microbial contamination is to be attributed to the personnel and no substantial microbial pitfalls in our Cell Factory has been detected.

Local control: a hub-based model for the c-di-GMP network.

The genome of Pseudomonas fluorescens encodes >50 proteins predicted to play a role in bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-mediated biofilm formation. We built a network representation of protein-protein interactions and extracted key information via multidimensional scaling (i.e., principal component analysis) of node centrality measures, which measure features of proteins in a network. Proteins of different domain types (diguanylate cyclase, dual domain, phosphodiesterase, PilZ) exhibit unique network behavior and can be accurately classified by their network centrality values (i.e., roles in the network). The predictive power of protein-protein interactions in biofilm formation indicates the possibility of localized pools of c-di-GMP. A regression model showed a statistically significant impact of protein-protein interactions on the extent of biofilm formation in various environments. These results highlight the importance of a localized c-di-GMP signaling, extend our understanding of signaling by this second messenger beyond the current "Bow-tie Model," support a newly proposed "Hub Model," and suggest future avenues of investigation.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

The expanding role of blood and tissue establishments in the development of advanced therapy medicinal products.

BACKGROUND AIMS: The relationship between blood establishments and advanced cellular therapies is evident in several European countries, with some involved in research and development and/or in manufacturing. The aim of the present study was to understand the advanced therapy medicinal product (ATMP) infrastructural, regulatory and logistic requirements needed for the Irish Blood Transfusion Service to support advanced therapeutics in Ireland. METHODS: An online survey consisting of 13 questions was distributed in a targeted manner to the identified ATMP stakeholders in Ireland, namely those working in industry, health care, regulatory agencies or education. Subject matter experts in the field were approached and interviewed to gain further insight into the relationship between blood and tissue establishments (BTEs) and ATMPs, to explore the advantages these institutions have in development and to highlight potential challenges for implementation. RESULTS: In total, 84.9% of survey respondents stated that BTEs have a role in the development of advanced therapeutics. Key BTE services identified as applicable to the ATMP sector from both surveys and interviews include the provision of starting materials for research and manufacturing, donor management, use of existing quality and traceability frameworks, product logistic strategies and Good Manufacturing Practice. Challenges for BTE expansion into the sector currently include high costs associated with ATMPs, lack of expertise in these therapies, limited therapeutic populations and no national ATMP strategic plan for Ireland. CONCLUSIONS: Blood establishments have services and expertise that can be extended into the advanced therapy sector. The existing knowledge and skill base of BTEs in Ireland should be leveraged to accelerate the development of ATMP strategies for industry and healthcare.

Implementation of good manufacturing practices (GMP) to improve the quality of smoked fish (Scombercolias).

For several years, fish smoking has been the widely adopted processing method among artisanal fish smokers located along the coastal zones in many parts of West Africa including Ghana. However, several issues pertaining to biochemical and microbiological contaminants still remain, mainly because of the suboptimal, unhygienic fish handling during the processing. To help curtail the problem, we developed and implemented a simple good manufacturing practice (GMP) system for experimentation at two local fish smoking facilities (Facility A, FA; Facility B, FB) to assess the effectiveness for improving the quality of smoked fish. The implementation of GMP did not affect the physical properties of the smoked fish but improved the peroxide value, total volatile base nitrogen, polyaromantic hydrocarbons and histamine levels. The total aerobic counts decreased from 3.96 ± 0.12 cfu/g to 1.52 ± 0.28 cfu/g (FA) or from 4.10 ± 0.2 cfu/g to 1.85 ± 0.85 cfu/g, (FB). The coliforms and Escherichia coli decreased respectively from 1.69 ± 0.12 cfu/g and 1.15 ± 0.21 cfu/g (FA) and from 1.74 ± 0.37 cfu/g and 1.24 ± 0.37 cfu/g, (FB) to below detection (no observed colony) after introducing the single use of potable water, use of smoking oven and fish core temperature of 108.1 ± 7.5 °C and 82.5 ± 3.9 °C, respectively for 2 h, wearing of safety apparels, drying and cooling of smoked fish under nets, and the use of waste disposal bins. The results show that sensitization and training of fish smokers in GMP may be relevant for improving the microbial and overall quality of smoked fish.

Biofilm formation of Hafnia paralvei induced by c-di-GMP through facilitating bcsB gene expression promotes spoilage of Yellow River carp (Cyprinus carpio).

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Dynamics-driven allosteric stimulation of diguanylate cyclase activity in a red light-regulated phytochrome.

Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.

Correlates of Plasma NT-proBNP/Cyclic GMP Ratio in Heart Failure With Preserved Ejection Fraction: An Analysis of the RELAX Trial.

BACKGROUND: Phosphodiesterases degrade cyclic GMP (cGMP), the second messenger that mediates the cardioprotective effects of natriuretic peptides. High natriuretic peptide/cGMP ratio may reflect, in part, phosphodiesterase activity. Correlates of natriuretic peptide/cGMP in patients with heart failure with preserved ejection fraction are not well understood. Among patients with heart failure with preserved ejection fraction in the RELAX (Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure With Preserved Ejection Fraction) trial, we examined (1) cross-sectional correlates of circulating NT-proBNP (N-terminal pro-B-type natriuretic peptide)/cGMP ratio, (2) whether selective phosphodiesterase-5 inhibition by sildenafil changed the ratio, and (3) whether the effect of sildenafil on 24-week outcomes varied by baseline ratio. METHODS AND RESULTS: In 212 subjects, NT-proBNP/cGMP ratio was calculated at randomization and 24 weeks. Correlates of the ratio and its change were examined in multivariable proportional odds models. Whether baseline ratio modified the sildenafil effect on outcomes was examined by interaction terms. Higher NT-proBNP/cGMP ratio was associated with greater left ventricular mass and troponin, the presence of atrial fibrillation, and lower estimated glomerular filtration rate and peak oxygen consumption. Compared with placebo, sildenafil did not alter the ratio from baseline to 24 weeks (P=0.17). The effect of sildenafil on 24-week change in peak oxygen consumption, left ventricular mass, or clinical composite outcome was not modified by baseline NT-proBNP/cGMP ratio (P-interaction >0.30 for all). CONCLUSIONS: Among patients with heart failure with preserved ejection fraction, higher NT-proBNP/cGMP ratio associated with an adverse cardiorenal phenotype, which was not improved by selective phosphodiesterase-5 inhibition. Other phosphodiesterases may be greater contributors than phosphodiesterase-5 to the adverse phenotype associated with a high natriuretic peptide/cGMP ratio in HFpEF. REGISTRATION INFORMATION: clinicaltrials.gov. Identifier: NCT00763867.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella.

Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

Optimization of the Production Process of Clinical-Grade Human Salivary Gland Organoid-Derived Cell Therapy for the Treatment of Radiation-Induced Xerostomia in Head and Neck Cancer.

Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline in amylase production. Currently, there is no curative treatment for radiation-induced hyposalivation/xerostomia. This study aimed to develop and optimize a validated manufacturing process for salivary gland organoid cells containing stem/progenitor cells using salivary gland patient biopsies as a starting material. The manufacturing process should comply with GMP requirements to ensure clinical applicability. A laboratory-scale process was further developed into a good manufacturing practice (GMP) process. Clinical-grade batches complying with set acceptance and stability criteria were manufactured. The results showed that the manufactured salivary gland-derived cells were able to self-renew, differentiate, and show functionality. This study describes the optimization of an innovative and promising novel cell-based therapy.

Methods for generating the CD137L-DC-EBV-VAX anti-cancer vaccine.

Dendritic cells (DC) are professional antigen presenting cells (APCs) that can efficiently present captured antigens to cytotoxic T cells and initiate powerful antigen-specific responses. Therefore, DC have been explored for cancer immunotherapy. However, due to the scarcity of DCs in the peripheral blood, ex-vivo expansion is required to generate sufficient DCs before use. The majority of DC-based tumor vaccines utilize monocyte-derived DC (mo-DC) that are generated with GM-CSF and IL-4. Here, we describe the generation of a novel type of DC, CD137L-DC, which are generated from monocytes by stimulation with a CD137 ligand agonist, and that proved to be more potent than classical mo-DC in inducing cytotoxic responses against tumor associated viruses, such as EBV and HBV in vitro. In a phase I clinical trial on patients with locally recurrent or metastatic NPC, a CD137L-DC-EBV vaccine showed good tolerability and prolonged patient survival, providing a basis for further development of CD137L-DC vaccines for immunotherapy.

EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

cGAS: action in the nucleus.

As a canonical cytoplasmic DNA sensor, cyclic GMP-AMP synthase (cGAS) plays a key role in innate immunity. In recent years, a growing number of studies have shown that cGAS can also be located in the nucleus and plays new functions such as regulating DNA damage repair, nuclear membrane repair, chromosome fusion, DNA replication, angiogenesis and other non-canonical functions. Meanwhile, the mechanisms underlying the nucleo-cytoplasmic transport and the regulation of cGAS activation have been revealed in recent years. Based on the current understanding of the structure, subcellular localization and canonical functions of cGAS, this review focuses on summarizing the mechanisms underlying nucleo-cytoplasmic transport, activity regulation and non-canonical functions of cGAS in the nucleus. We aim to provide insights into exploring the new functions of cGAS in the nucleus and advance its clinical translation.

Optimization of anti-CD19 CAR T cell production for treatment of patients with chronic lymphocytic leukemia.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) have activity against chronic lymphocytic leukemia (CLL), but complete response rates range from 18% to 29%, so improvement is needed. Peripheral blood mononuclear cells (PBMCs) of CLL patients often contain high levels of CLL cells that can interfere with CAR T cell production, and T cells from CLL patients are prone to exhaustion and other functional defects. We previously developed an anti-CD19 CAR designated Hu19-CD828Z. Hu19-CD828Z has a binding domain derived from a fully human antibody and a CD28 costimulatory domain. We aimed to develop an optimized process for producing Hu19-CD828Z-expressing T cells (Hu19-CAR T) from PBMC of CLL patients. We determined that supplementing Hu19-CAR-T cultures with interleukin (IL)-7 + IL-15 had advantages over using IL-2, including greater accumulation of Hu19-CAR T cells during in vitro proliferation assays. We determined that positive selection with anti-CD4 and anti-CD8 magnetic beads was the optimal method of T cell purification because this method resulted in high T cell purity. We determined that anti-CD3/CD28 paramagnetic beads were the optimal T cell activation reagent. Finally, we developed a current good manufacturing practices-compliant clinical-scale protocol for producing Hu19-CAR T from PBMC of CLL patients. These Hu19-CAR T exhibited a full range of in vitro functions and eliminated leukemia from mice.