Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

TLR9 and STING agonists cooperatively boost the immune response to SARS-CoV-2 RBD vaccine through an increased germinal center B cell response and reshaped T helper responses.

Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.

Repurposing Astragalus Polysaccharide PG2 for Inhibiting ACE2 and SARS-CoV-2 Spike Syncytial Formation and Anti-Inflammatory Effects.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to global public health. In an effort to develop novel anti-coronavirus therapeutics and achieve prophylactics, we used gene set enrichment analysis (GSEA) for drug screening and identified that Astragalus polysaccharide (PG2), a mixture of polysaccharides purified from Astragalus membranaceus, could effectively reverse COVID-19 signature genes. Further biological assays revealed that PG2 could prevent the fusion of BHK21-expressing wild-type (WT) viral spike (S) protein and Calu-3-expressing ACE2. Additionally, it specifically prevents the binding of recombinant viral S of WT, alpha, and beta strains to ACE2 receptor in our non-cell-based system. In addition, PG2 enhances let-7a, miR-146a, and miR-148b expression levels in the lung epithelial cells. These findings speculate that PG2 has the potential to reduce viral replication in lung and cytokine storm via these PG2-induced miRNAs. Furthermore, macrophage activation is one of the primary issues leading to the complicated condition of COVID-19 patients, and our results revealed that PG2 could regulate the activation of macrophages by promoting the polarization of THP-1-derived macrophages into an anti-inflammatory phenotype. In this study, PG2 stimulated M2 macrophage activation and increased the expression levels of anti-inflammatory cytokines IL-10 and IL-1RN. Additionally, PG2 was recently used to treat patients with severe COVID-19 symptoms by reducing the neutrophil-to-lymphocyte ratio (NLR). Therefore, our data suggest that PG2, a repurposed drug, possesses the potential to prevent WT SARS-CoV-2 S-mediated syncytia formation with the host cells; it also inhibits the binding of S proteins of WT, alpha, and beta strains to the recombinant ACE2 and halts severe COVID-19 development by regulating the polarization of macrophages to M2 cells.

Functional Characterization and Structural Modeling of a Novel Glycine Oxidase from Variovorax paradoxus Iso1.

The N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of N-acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the N-d-AAase gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl ß-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and N-d-AAase were expressed on the same mRNA and could be transcriptionally induced by various N-acetyl-d-amino acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with N-d-AAase in an operon, and metabolizes N-acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. IMPORTANCE The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an N-acyl-d-amino-acid amidohydrolase (N-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both in vitro and in vivo) another potential gene found in the promoter region of this N-d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with N-d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology.

Virofree, an Herbal Medicine-Based Formula, Interrupts the Viral Infection of Delta and Omicron Variants of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) remains a threat with the emergence of new variants, especially Delta and Omicron, without specific effective therapeutic drugs. The infection causes dysregulation of the immune system with a cytokine storm that eventually leads to fatal acute respiratory distress syndrome (ARDS) and further irreversible pulmonary fibrosis. Therefore, the promising way to inhibit infection is to disrupt the binding and fusion between the viral spike and the host ACE2 receptor. A transcriptome-based drug screening platform has been developed for COVID-19 to explore the possibility and potential of the long-established drugs or herbal medicines to reverse the unique genetic signature of COVID-19. In silico analysis showed that Virofree, an herbal medicine, reversed the genetic signature of COVID-19 and ARDS. Biochemical validations showed that Virofree could disrupt the binding of wild-type and Delta-variant spike proteins to ACE2 and its syncytial formation via cell-based pseudo-typed viral assays, as well as suppress binding between several variant recombinant spikes to ACE2, especially Delta and Omicron. Additionally, Virofree elevated miR-148b-5p levels, inhibited the main protease of SARS-CoV-2 (Mpro), and reduced LPS-induced TNF-α release. Virofree also prevented cellular iron accumulation leading to ferroptosis which occurs in SARS-CoV-2 patients. Furthermore, Virofree was able to reduce pulmonary fibrosis-related protein expression levels in vitro. In conclusion, Virofree was repurposed as a potential herbal medicine to combat COVID-19. This study highlights the inhibitory effect of Virofree on the entry of Delta and Omicron variants of SARS-CoV-2, which have not had any effective treatments during the emergence of the new variants spreading.

Rapid microfluidic platform for screening and enrichment of cells secreting virus neutralizing antibodies.

As part of the body's immune response, antibodies (Abs) have the ability to neutralize pathogenic viruses to prevent infection. To screen for neutralizing Abs (nAbs) from the immune repertoire, multiple screening techniques have been developed. However, conventional methods have a trade-off between screening throughput and the ability to screen for nAbs via their functional efficacy. Although droplet microfluidic platforms have the ability to bridge this disparity, the majority of such reported platforms still rely on Ab-binding assays as a proxy for function, which results in irrelevant hits. Herein, we report the multi-module Droplet-based Platform for Effective Antibody RetrievaL (DROP-PEARL) platform, which can achieve high-throughput enrichment of Ab-secreting cells (ASCs) based on the neutralizing activity of secreted nAbs against the a target virus. In this study, in-droplet Chikungunya virus (CHIKV) infection of host cells and neutralization was demonstrated via sequential delivery of viruses and host cells via picoinjection. In addition, we demonstrate the ability of the sorting system to accurately discriminate and isolate uninfected droplets from a mixed population of droplets at a rate of 150 000 cells per hour. As a proof of concept, a single-cell neutralization assay was performed on two populations of cells (nAb-producing and non-Ab producing cells), and up to 2.75-fold enrichment of ASCs was demonstrated. Finally, we demonstrated that DROP-PEARL is able to achieve similar enrichment for low frequency (∼2%) functional nAb-producing cells in a background of excess cells secreting irrelevant antibodies, highlighting its potential prospect as a first round enrichment platform for functional ASCs. We envision that the DROP-PEARL platform could potentially be used to accelerate the discovery of nAbs against other pathogenic viral targets, and we believe it will be a useful in the ongoing fight against biological threats.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Sensitive detection of total anti-Spike antibodies and isotype switching in asymptomatic and symptomatic individuals with COVID-19.

Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.

Quorum-Sensing Regulator OpaR Directly Represses Seven Protease Genes in Vibrio parahaemolyticus.

Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.

An Analysis of Isoclonal Antibody Formats Suggests a Role for Measuring PD-L1 with Low Molecular Weight PET Radiotracers.

PURPOSE: The swell of new and diverse radiotracers to predict or monitor tumor response to cancer immunotherapies invites the opportunity for comparative studies to identify optimal platforms. To probe the significance of antibody format on image quality for PD-L1 imaging, we developed and studied the biodistribution of a library of antibodies based on the anti-PD-L1 IgG1 clone C4. PROCEDURE: A C4 minibody and scFv were cloned, expressed, and characterized. The antibodies were functionalized with desferrioxamine and radiolabeled with Zr-89 to enable a rigorous comparison with prior data collected using 89Zr-labeled C4 IgG1. The biodistribution of the radiotracers was evaluated in C57Bl6/J or nu/nu mice bearing B16F10 or H1975 tumors, respectively, which are models that represent high and low tumor autonomous PD-L1 expression. RESULTS: The tumor uptake of the 89Zr-C4 minibody was higher than 89Zr-C4 scFv and equivalent to previous data collected using 89Zr-C4 IgG1. However, the peak tumors to normal tissue ratios were generally higher for 89Zr-C4 scFv compared with 89Zr-C4 minibody and 89Zr-IgG1. Moreover, an exploratory study showed that the rapid clearance of 89Zr-C4 scFv enabled detection of endogenous PD-L1 on a genetically engineered and orthotopic model of hepatocellular carcinoma. CONCLUSION: In summary, these data support the use of low molecular weight constructs for PD-L1 imaging, especially for tumor types that manifest in abdominal organs that are obstructed by the clearance of high molecular weight radioligands.

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization.

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Characterization of a Phosphatidylserine Synthase of Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.

Knowledge, Attitude and Behaviours Related to Weight Control and Body-Image Perceptions among Chinese High School Students.

BACKGROUND: Body-image perception is one of the determinants in weight management, especially among adolescents. This study aimed to assess weight-control knowledge, attitude and behaviours along with body-image perceptions among Chinese high school students in order to compare the weight-control behaviours with those perceptions. METHODS: A cross-sectional study was conducted among 277 Chinese students in Form 1 and Form 2 in Pulau Pinang, Malaysia using convenience sampling. The following outcomes were evaluated: weight-control knowledge, attitude and behaviours (weight-related knowledge and attitude questionnaire; weight control strategies scale) and body-image perceptions (figure rating scale). RESULTS: Both genders were found to have high weight-control knowledge, with female adolescents scoring significantly higher than male adolescents (P = 0.010). However, only half of the adolescents (50.9%) perceived that obesity is bad for health. Although only 44.4% of adolescents were dissatisfied with their current weight status, 62.8% intended to change their present weight status. Male adolescents significantly engaged more in physical activity (PA) (P = 0.035) and self-monitoring (SM) (P = 0.014) compared to their female counterparts. Furthermore, male adolescents chose their current body size as their ideal body image, but female adolescents preferred a slimmer ideal figure. The percentage of male and female adolescents who desired a smaller body figure was 39.6% and 54.5%, respectively. Lastly, there was no significant difference between weight-control behaviours and adolescents' body-image perceptions. CONCLUSION: Female Chinese adolescents had higher weight-control knowledge and preferred a slimmer body size, yet males were more likely to engage in PA and SM behaviours. Essentially, imprecise attitude towards obesity among half of the Chinese high school students is of particular concern.

Molecular and functional evidence of phosphatidylserine synthase in Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Enhancing Antigen Cross-Presentation in Human Monocyte-Derived Dendritic Cells by Recruiting the Intracellular Fc Receptor TRIM21.

Suboptimal immune responses to pathogens contribute to chronic infections. One way to improve immune responses is to boost Ag presentation. In this study, we investigate the potential of the tripartite motif-containing 21 (TRIM21) pathway. TRIM21 is a ubiquitously expressed cytosolic protein that recognizes the Fc region of Abs. When Abs that are bound to pathogens enter the cell as immune complexes, binding of TRIM21 to Fc initiates downstream inflammatory signaling and targets the immune complexes for proteasomal degradation. In APCs, peptides generated by proteasomes are loaded onto MHC class I molecules to stimulate CD8 T cell responses, which are crucial for effective immunity to pathogens. We hypothesized that increasing the affinity between immune complexes and TRIM21 might markedly improve CD8 T cell responses to Ags processed by the TRIM21 pathway. Using phage display technology, we engineered the human IgG1 Fc to increase its affinity for TRIM21 by 100-fold. Adenovirus immune complexes with the engineered Fc induced greater maturation of human dendritic cells (DC) than immune complexes with unmodified Fc and stimulated increased Ag-specific CD8 T cell proliferation and IFN-γ release in cocultures of DC-PBMC. Thus, by increasing the affinity between Fc and TRIM21, Ags from immune complexes undergo enhanced cross-presentation on DC, leading to greater CD8 T cell responses. Our study reveals an approach that could potentially be used in vaccines to increase cytotoxic T cell responses against Ags that are targeted or delivered by Fc-modified Abs.

A Preclinical Assessment of 89Zr-atezolizumab Identifies a Requirement for Carrier Added Formulations Not Observed with 89Zr-C4.

The swell of experimental imaging technologies to noninvasively measure immune checkpoint protein expression presents the opportunity for rigorous comparative studies toward identifying a gold standard. 89Zr-atezolizumab is currently in man, and early data show tumor targeting but also abundant uptake in several normal tissues. Therefore, we conducted a reverse translational study both to understand if tumor to normal tissue ratios for 89Zr-atezolizumab could be improved and to make direct comparisons to 89Zr-C4, a radiotracer that we showed can detect a large dynamic range of tumor-associated PD-L1 expression. PET/CT and biodistribution studies in tumor bearing immunocompetent and nu/nu mice revealed that high specific activity 89Zr-atezolizumab (∼2 µCi/µg) binds to PD-L1 on tumors but also results in very high uptake in many normal mouse tissues, as expected. Unexpectedly, 89Zr-atezolizumab uptake was generally higher in normal mouse tissues compared to 89Zr-C4 and lower in H1975, a tumor model with modest PD-L1 expression. Also unexpectedly, reducing the specific activity at least 15-fold suppressed 89Zr-atezo uptake in normal mouse tissues but increased tumor uptake to levels observed with high specific activity 89Zr-C4. In summary, these data reveal that low specific activity 89Zr-atezo may be necessary for accurately measuring PD-L1 in the tumor microenvironment, assuming a threshold can be identified that preferentially suppresses binding in normal tissues without reducing binding to tumors with abundant expression. Alternatively, high specific activity approaches like 89Zr-C4 PET may be simpler to implement clinically to measure the broad dynamic range of PD-L1 expression known to manifest among tumors.

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol.

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L-1 h-1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.

Imaging PD-L1 Expression with ImmunoPET.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

OpaR and RpoS are positive regulators of a virulence factor PrtA in Vibrio parahaemolyticus.

PrtA is an extracellular serine protease of Vibrio parahaemolyticus and has haemolytic and cytotoxic activities. Many extracellular proteases have been shown to be required for nutrient intake and the infection mechanism of vibrios. In this study, we report that OpaR, a quorum sensing regulator, and RpoS, a general stress response regulator, play important roles in the PrtA regulation pathway. Extracellular protease activity was highest during the late-log growth of Vibrio parahaemolyticus no.93 (VP93). The absence of PrtA distinctly decreased the extracellular protease activity. Deletion of opaR or rpoS alone reduced PrtA-specific activity of VP93. Quantitative reverse-transcriptase PCR and Western blot analysis suggested that OpaR and RpoS promote PrtA expression at the transcriptional level and affect the amount of extracellular PrtA. A luciferase assay revealed that OpaR regulates prtA on the prtA promoter region. Electrophoretic mobility shift assays indicated that the purified His-OpaR was able to bind specifically to two sequences (PrtA-1 and PrtA-2) of the prtA promoter region. Footprinting analysis showed that OpaR regulates prtA by binding to the promoter region of prtA at positions -269 to -246 and -88 to -68 from the prtA translational start site. Together, the results suggest that PrtA was upregulated by two global regulators, OpaR and RpoS.