Inhibition of glycolysis enhances the efficacy of immunotherapy via PDK-mediated upregulation of PD-L1.

BACKGROUND: Immunotherapy for gastric cancer remains a challenge due to its limited efficacy. Metabolic reprogramming toward glycolysis has emerged as a promising avenue for enhancing the sensitivity of tumors to immunotherapy. Pyruvate dehydrogenase kinases (PDKs) play pivotal roles in regulating glycolysis. The importance of PDKs in the context of gastric cancer immunotherapy and their potential as therapeutic targets have not been fully explored. METHODS: PDK and PD-L1 expression was analyzed using data from the GSE66229 and The Cancer Genome Atlas (TCGA) cohorts. Additionally, the Immune Checkpoint Blockade Therapy Atlas (ICBatlas) database was utilized to assess PDK expression in an immune checkpoint blockade (ICB) therapy group. Subsequently, the upregulation of PD-L1 and the enhancement of anticancer effects achieved by targeting PDK were validated through in vivo and in vitro assays. The impact of PDK on histone acetylation was investigated using ChIP‒qPCR to detect changes in histone acetylation levels. RESULTS: Our analysis revealed a notable negative correlation between PD-L1 and PDK expression. Downregulation of PDK led to a significant increase in PD-L1 expression. PDK inhibition increased histone acetylation levels by promoting acetyl-CoA generation. The augmentation of acetyl-CoA production and concurrent inhibition of histone deacetylation were found to upregulate PD-L1 expression in gastric cancer cells. Additionally, we observed a significant increase in the anticancer effect of PD-L1 antibodies following treatment with a PDK inhibitor. CONCLUSIONS: Downregulation of PDK in gastric cancer cells leads to an increase in PD-L1 expression levels, thus potentially improving the efficacy of PD-L1 immune checkpoint blockade therapy.

SLC44A2 regulates vascular smooth muscle cell phenotypic switching and aortic aneurysm.

Aortic aneurysm is a life-threatening disease with limited interventions, closely related to vascular smooth muscle cells (VSMCs) phenotypic switching. SLC44A2, a member of solute carrier series 44 (SLC44) family, remains under-characterized in the context of cardiovascular diseases. Venn diagram analysis based on microarray and single-cell RNA sequencing identified SLC44A2 as a major regulator of VSMCs phenotypic switching in aortic aneurysm. Screening for Slc44a2 amongst aortic cell lineages demonstrated its predominant location in VSMCs. Elevated levels of SLC44A2 were evidenced in the aorta of both abdominal aortic aneurysm patients and angiotensin II (Ang II)-infused Apoe-/- mice. In vitro, SLC44A2 silencing promoted VSMCs towards a synthetic phenotype, while SLC44A2 overexpression attenuated VSMCs phenotypic switching. VSMCs-specific SLC44A2 knockout mice were more susceptible to aortic aneurysm under Ang II infusion, while SLC44A2 overexpression showed protective effects. Mechanistically, SLC44A2 interaction with NRP1 and ITGB3 activates TGF-ß/SMAD signaling, thereby promoting contractile genes expression. Elevated SLC44A2 in aortic aneurysm is associated with upregulated runt-related transcription factor 1 (RUNX1). Furthermore, low dose of lenalidomide (LEN) suppressed aortic aneurysm progression by enhancing SLC44A2 expression. These findings reveal SLC44A2/NRP1/ITGB3 complex is a major regulator of VSMCs phenotypic switching and provide potential therapeutic approach (LEN) for aortic aneurysm treatment.

Mixed reality guided root canal therapy.

Root canal therapy (RCT) is a widely performed procedure in dentistry, with over 25 million individuals undergoing it annually. This procedure is carried out to address inflammation or infection within the root canal system of affected teeth. However, accurately aligning CT scan information with the patient's tooth has posed challenges, leading to errors in tool positioning and potential negative outcomes. To overcome these challenges, a mixed reality application is developed using an optical see-through head-mounted display (OST-HMD). The application incorporates visual cues, an augmented mirror, and dynamically updated multi-view CT slices to address depth perception issues and achieve accurate tooth localization, comprehensive canal exploration, and prevention of perforation during RCT. Through the preliminary experimental assessment, significant improvements in the accuracy of the procedure are observed. Specifically, with the system the accuracy in position was improved from 1.4 to 0.4 mm (more than a 70% gain) using an Optical Tracker (NDI) and from 2.8 to 2.4 mm using an HMD, thereby achieving submillimeter accuracy with NDI. 6 participants were enrolled in the user study. The result of the study suggests that the average displacement on the crown plane of 1.27 ± 0.83 cm, an average depth error of 0.90 ± 0.72 cm and an average angular deviation of 1.83 ± 0.83°. Our error analysis further highlights the impact of HMD spatial localization and head motion on the registration and calibration process. Through seamless integration of CT image information with the patient's tooth, our mixed reality application assists dentists in achieving precise tool placement. This advancement in technology has the potential to elevate the quality of root canal procedures, ensuring better accuracy and enhancing overall treatment outcomes.

Development of a novel fluorescent protein-based probe for efficient detection of Pb2+ in serum inspired by the metalloregulatory protein PbrR691.

BACKGROUND: The accurate and rapid detection of blood lead concentration is of paramount importance for assessing human lead exposure levels. Fluorescent protein-based probes, known for their high detection capabilities and low toxicity, are extensively used in analytical sciences. However, there is currently a shortage of such probes designed for ultrasensitive detection of Pb2+, and no reported probes exist for the quantitative detection of Pb2+ in blood samples. This study aims to fill this critical void by developing and evaluating a novel fluorescent protein-based probe that promises accurate and rapid lead quantification in blood. RESULTS: A simple and small-molecule fluorescent protein-based probe was successfully constructed herein using a peptide PbrBD designed for Pb2+ recognition coupled to a single fluorescent protein, sfGFP. The probe retains a three-coordinate configuration to identify Pb2+ and has a high affinity for it with a Kd' of 1.48 ± 0.05 × 10-17 M. It effectively transfers the conformational changes of the peptide to the chromophore upon Pb2+ binding, leading to fast fluorescence quenching and a sensitive response to Pb2+. The probe offers a broad dynamic response range of approximately 37-fold and a linear detection range from 0.25 nM to 3500 nM. More importantly, the probe can resist interference of metal ions in living organisms, enabling quantitative analysis of Pb2+ in the picomolar to millimolar range in serum samples with a recovery percentage of 96.64%-108.74 %. SIGNIFICANCE: This innovative probe, the first to employ a single fluorescent protein-based probe for ultrasensitive and precise analysis of Pb2+ in animal and human serum, heralds a significant advancement in environmental monitoring and public health surveillance. Furthermore, as a genetically encoded fluorescent probe, this probe also holds potential for the in vivo localization and concentration monitoring of Pb2+.

Pancreatic Extragastrointestinal Stromal Tumor: A Case Report.

Gastrointestinal stromal tumors (GISTs) are soft tissue sarcomas that originate from the mesenchymal cells of the gastrointestinal tract. Extra-GISTs (EGISTs) are caused by sites outside the gastrointestinal tract. We reported a case of EGIST of the pancreas in a 51-year-old woman. Enhanced CT scan showed a rounded, slightly hypointense focus in the head of the pancreas and the right pars compacta of the descending duodenum. Routine laboratory and endocrine tests were unremarkable. The patient underwent laparoscopic surgery. The diagnosis of EGIST was confirmed through histopathological and immunohistochemical examination. The tumor was found to be CD117+, CD34+, and DOG+ with a high risk of malignancy. No recurrence was observed during the nine-month postoperative follow-up.

Establishment of an iPSC line (BCHNDi001-A) from a patient with nicotinamide nucleotide repair system deficiency caused by biallelic NAXD mutations.

NAD(P)HX dehydratase (NAXD) gene is one of the key enzymes encoding the nicotinamide nucleotide repair system, reportedly associated with Encephalopathy, progressive, early-onset, with brain edema and/or leukoencephalopathy, 2 (PEBEL2). Here, we generated an induced pluripotent stem cell (iPSC) line from the dermal fibroblasts (HDFs) of a PEBEL2 patient who carried biallelic mutations, c.101_102delTA(p.Thr35Phefs*63) and c.318C > G (p.Ile160Met) in NAXD. These iPSCs showed stable amplification in vitro, expressed pluripotent markers, and differentiated spontaneously into three germ layers, as well as NAXD mutations with normal karyotype.

Epigenetic regulation of CD38/CD48 by KDM6A mediates NK cell response in multiple myeloma.

Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Long-term effectiveness and tolerability of ketogenic diet therapy in patients with genetic developmental and epileptic encephalopathy onset within the first 6 months of life.

OBJECTIVE: To investigate the effectiveness and tolerability of ketogenic diet therapy (KDT) in patients with developmental and epileptic encephalopathy (DEE) associated with genetic etiology which onset within the first 6 months of life, and to explore the association between response to KDT and genotype/clinical parameters. METHODS: We retrospectively reviewed data from patients with genetic DEE who started KDT at Beijing Children's Hospital between January 1, 2016, and December 31, 2021. RESULTS: A total of 32 patients were included, involving 14 pathogenic or likely pathogenic single genes, and 16 (50.0%) patients had sodium/potassium channel gene variants. The median age at onset of epilepsy was 1.0 (IQR: 0.1, 3.0) months. The median age at initiation of KDT was 10.0 (IQR: 5.3, 13.8) months and the median duration of maintenance was 14.0 (IQR: 7.0, 26.5) months, with a mean blood ß-hydroxybutyrate of 2.49 ± 0.62 mmol/L. During the maintenance period of KDT, 26 (81.3%) patients had a ≥50% reduction of seizure frequency, of which 12 (37.5%) patients achieved seizure freedom. Better responses were observed in patients with STXBP1 variants, with four out of five patients achieving seizure freedom. There were no statistically differences in the age of onset, duration of epilepsy before KDT, blood ketone values, or the presence of ion channel gene variants between the seizure-free patients and the others. The most common adverse effects were gastrointestinal side effects, which occurred in 21 patients (65.6%), but all were mild and easily corrected. Only one patient discontinued KDT due to nephrolithiasis. SIGNIFICANCE: KDT is effective in treating early onset genetic DEE, and no statistically significant relationship has been found between genotype and effectiveness in this study. KDT is well tolerated in most young patients, with mild and reversible gastrointestinal side effects being the most common, but usually not the reason to discontinue KDT. PLAIN LANGUAGE SUMMARY: This study evaluated the response and side effects of ketogenic diet therapy (KDT) in patients who had seizures within the first 6 months of life, and were diagnosed with genetic developmental and epileptic encephalopathy (DEE), a type of severe epilepsy with developmental delay caused by gene variants. Thirty-two patients involving 14 gene variants who started KDT at Beijing Children's Hospital between were included. KDT was effective in treating early onset genetic DEE in this cohort, and patients with STXBP1 variants responded better; however, no statistically significant relationship was found between gene variant and response. Most young patients tolerated KDT well, with mild and reversible gastrointestinal side effects being the most common.

First Display of Haloalkane Dehalogenase LinB on the Surface of Bacillus subtilis Spore.

BACKGROUND: LinB, as a Haloalkane dehalogenase, has good catalytic activity for many highly toxic and recalcitrant compounds, and can realize the elimination of chemical weapons HD in a green non-toxic mode. OBJECTIVES: In order to display Haloalkane dehalogenase LinB on the surface of Bacillus subtilis spore. METHODS: We have constituted the B. subtilis spore surface display system of halogenated alkanes dehalogenase LinB by gene recombination. RESULTS: Data revealed that LinB can display on spore surface successfully. The hydrolyzing HD analogue 2-chloroethyl ethylsulfide (2-CEES) activity of displayed LinB spores was 4.30±0.09 U/mL, and its specific activity was 0.78±0.03U/mg. Meanwhile, LinB spores showed a stronger stress resistance activity on 2-CEES than free LinB. This study obtained B. subtilis spores of LinB (phingobium japonicum UT26) with enzyme activity that was not reported before. CONCLUSION: Spore surface display technology uses resistance spore as the carrier to guarantee LinB activity, enhances its stability, and reduces the production cost, thus expanding the range of its application.

Systematic profiling of conditional pathway activation identifies context-dependent synthetic lethalities.

The paradigm of cancer-targeted therapies has focused largely on inhibition of critical pathways in cancer. Conversely, conditional activation of signaling pathways as a new source of selective cancer vulnerabilities has not been deeply characterized. In this study, we sought to systematically identify context-specific gene-activation-induced lethalities in cancer. To this end, we developed a method for gain-of-function genetic perturbations simultaneously across ~500 barcoded cancer cell lines. Using this approach, we queried the pan-cancer vulnerability landscape upon activating ten key pathway nodes, revealing selective activation dependencies of MAPK and PI3K pathways associated with specific biomarkers. Notably, we discovered new pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct ß-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. Together, this study reveals a new class of conditional gene-activation dependencies in cancer.

Thiamine pyrophosphokinase deficiency: report of two Chinese cases and a literature review.

Thiamine pyrophosphokinase (TPK) deficiency, is a rare autosomal recessive disorder of congenital metabolic dysfunction caused by variants in the TPK1 gene. TPK1 variants can lead to thiamine metabolic pathway obstacles, and its clinical manifestations are highly variable. We describe two cases of TPK deficiency with completely different phenotypes and different therapeutic effects, and 26 cases of previously reported were retrospectively reviewed to improve our understanding of the clinical and genetic features of the disease. Patients with TPK deficiency present with ataxia, dysarthria, dystonia, disturbance of consciousness, seizures, and other nervous system dysfunction. Different gene variant sites may lead to different clinical features and therapeutic effects. Gene analysis is important for the diagnosis of TPK deficiency caused by TPK1 variants, and thiamine supplementation has been the mainstay of treatment for TPK deficiency to date.

Efflux transport proteins of Tetrahymena thermophila play important roles in resistance to perfluorooctane sulfonate exposure.

The biotoxicity of perfluorooctane sulfonate (PFOS) has been a concern. However, the effects of PFOS on Tetrahymena thermophila, a unicellular model organism, remain unclear. This study aimed to investigate the toxicity and detoxification mechanism of PFOS in this protozoan. PFOS did not show prominent toxic effects on T. thermophila. Cell viability of T. thermophila can be concentration-dependently increased by PFOS. PFOS also increased the stability of cell membranes and the activity of lysosomes. However, PFOS inhibited efflux transporter activities. Most of the PFOS amount remained in the culture medium during the culture periods. Only a low amount of PFOS was absorbed by cells, where PFOS molecules were mainly combined with membrane proteins. The expressions of four membrane protein genes involved in transporting xenobiotics were analyzed by real time-PCR. The gene abcg25 was significantly up-regulated. The growth of abcg25 gene knockout protozoans under PFOS treatment was slightly inhibited. However, the amount of PFOS adsorbed by the knockout protozoans showed no significant difference from the Wild-type protozoans. We concluded that the ABCG25 protein might play a key role in preventing PFOS from entering the cell or being exported from the cells to protect T. thermophila against PFOS. However, ABCG25 was not the only membrane protein able to bind with PFOS.

Identification of a (+)-cubenene synthase from filamentous fungi Acremonium chrysogenum.

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.

STTAR: Surgical Tool Tracking using Off-the-Shelf Augmented Reality Head-Mounted Displays.

The use of Augmented Reality (AR) for navigation purposes has shown beneficial in assisting physicians during the performance of surgical procedures. These applications commonly require knowing the pose of surgical tools and patients to provide visual information that surgeons can use during the performance of the task. Existing medical-grade tracking systems use infrared cameras placed inside the Operating Room (OR) to identify retro-reflective markers attached to objects of interest and compute their pose. Some commercially available AR Head-Mounted Displays (HMDs) use similar cameras for self-localization, hand tracking, and estimating the objects' depth. This work presents a framework that uses the built-in cameras of AR HMDs to enable accurate tracking of retro-reflective markers without the need to integrate any additional electronics into the HMD. The proposed framework can simultaneously track multiple tools without having previous knowledge of their geometry and only requires establishing a local network between the headset and a workstation. Our results show that the tracking and detection of the markers can be achieved with an accuracy of 0.09±0.06 mm on lateral translation, 0.42 ±0.32 mm on longitudinal translation and 0.80 ±0.39° for rotations around the vertical axis. Furthermore, to showcase the relevance of the proposed framework, we evaluate the system's performance in the context of surgical procedures. This use case was designed to replicate the scenarios of k-wire insertions in orthopedic procedures. For evaluation, seven surgeons were provided with visual navigation and asked to perform 24 injections using the proposed framework. A second study with ten participants served to investigate the capabilities of the framework in the context of more general scenarios. Results from these studies provided comparable accuracy to those reported in the literature for AR-based navigation procedures.

A novel strategy of engineering genetically encoded probe for ultrasensitive sensing Hg2+ with unusual planar trigonometric coordination configuration.

At present, few genetically encoded fluorescent probes are currently available for the analysis of toxic heavy metal ions, and most have poor performance that cannot meet the requirements of sensitive and dynamic detection in living cells. In this study, we designed a single fluorescent protein-based probe sfGFP-MerBD, which can specifically response to Hg2+ with high binding affinity and wide dynamic range. More importantly, the developing probe can timely and reversibly monitor changes of Hg2+ concentration in living mammalian cells. The excellent performance of this probe is largely due to the recognition element of the probe, MerBD, which adopts an unusual planar trigonometric coordination configuration with Hg2+, and the coordination can cause enough conformational change to influence the fluorescence of skeleton protein sfGFP coupled with it. The small peptide MerBD was delicately designed based on the three-dimensional structure of metalloprotein MerR. This novel design strategy solves the challenging problems that there are few natural functional proteins in the process of constructing fluorescent probes for toxic metal ions and some functional proteins cannot be directly used as recognition elements. Based on the new strategy, more genetically encoded fluorescent probes of toxic heavy metal ions can be efficiently constructed and applied in the future.

A novel, genetically encoded whole-cell biosensor for directed evolution of myrcene synthase in Escherichia coli.

ß-myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite.

Transcriptomic analysis of polyketide synthesis in dinoflagellate, Prorocentrum lima.

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

Analysis of the current status and influencing factors of cross-regional hospitalization services utilization by basic medical insurance participants in China - taking a central province as an example.

Background: The geographically uneven distribution of healthcare resources has resulted in a dramatic increase of cross-regional hospitalization services in China. The over-use of cross-regional hospitalization services may hinder the utilization and improvement of local hospitalization services. It is of great practical significance to study the utilization of cross-regional hospitalization services and its influencing factors in order to effectively allocate medical resources and guide patients to seek medical treatment rationally. Therefore, this study aims to analyze the current situation and influencing factors of the utilization of cross-regional hospitalization services by patients insured by basic medical insurance in China. Methods: A total of 3,291 cross-provincial inpatients were randomly selected in a central province of China in 2020. The level of medical institutions, hospitalization expenses and actual reimbursement rate were selected as indicators of hospitalization service utilization. Exploratory factor analysis was used to assess the dimensionality of influencing factors and reduce the number of variables, and binomial logistic regression analysis and multiple linear regression analysis to explore the influencing factors of the utilization of cross-regional hospitalization services. Results: The proportion of cross-provincial inpatients choosing tertiary hospitals was the highest with average hospitalization expenses of 24,662 yuan and an actual reimbursement rate of 51.0% on average. Patients insured by Urban Employees' Basic Medical Insurance (UEBMI) were more frequently (92.9% vs. 88.5%) to choose tertiary hospitals than those insured by Urban and Rural Residents' Basic Medical Insurance (URRBMI), and their average hospitalization expenses (30,727 yuan) and actual reimbursement rate (68.2%) were relatively higher (p < 0.001). The factor "income and security," "convenience of medical treatment" and "disease severity" had significant effects on inpatients' selection of medical institution level, hospitalization expenses and actual reimbursement rate, while the factor "demographic characteristics" only had significant effects on hospitalization expenses and actual reimbursement rate. Conclusion: Cross-provincial inpatients choose tertiary hospitals more frequently, and their financial burdens of medical treatment are heavy. A variety of factors jointly affect the utilization of cross-provincial hospitalization services for insured patients. It is necessary to narrow down the gap of medical treatment between UEBMI and URRBMI patients, and make full use of high-quality medical resources across regions.