New radiofrequency ablation procedure for selective reduction in complicated monochorionic twin or triplet pregnancy using multistep, incremental expansion technique.

OBJECTIVE: Radiofrequency ablation (RFA) is the preferred approach for selective reduction in complex monochorionic (MC) multiple pregnancies owing to the ease of operation and minimal invasiveness. To optimize the RFA technique and reduce the risk of adverse pregnancy outcome resulting from the heat-sink effect of RFA therapy, we used an innovative RFA method, in which an electrode needle was expanded incrementally and stepwise. This study aimed to assess the efficacy and safety profile of this novel multistep incremental expansion RFA method for selective fetal reduction in MC twin and triplet pregnancies. METHODS: This was a single-center retrospective cohort study of all MC multiple pregnancies undergoing RFA between March 2016 and October 2022 at our center. The multistep RFA technique involved the use of an expandable needle, which was gradually expanded during the RFA procedure until cessation of umbilical cord blood flow was achieved. The needle used for the single-step RFA method was fully extended from the start of treatment. RESULTS: In total, 132 MC multiple pregnancies underwent selective reduction using RFA, including 50 cases undergoing multistep RFA and 82 cases undergoing single-step RFA. The overall survival rates were not significantly different between the multistep and single-step RFA groups (81.1% vs 72.3%; P = 0.234). Similarly, the rates of preterm prelabor rupture of the membranes within 2 weeks after RFA, procedure-related complications, spontaneous preterm delivery and pathological findings on cranial ultrasound, as well as gestational age at delivery and birth weight, did not differ between the two groups. However, there was a trend towards a prolonged procedure-to-delivery interval following multistep RFA compared with single-step RFA (median, 109 vs 99 days; P = 0.377). Moreover, the fetal loss rate within 2 weeks after RFA in the multistep RFA group was significantly lower than that in the single-step RFA group (10.0% vs 24.4%; P = 0.041). The median ablation time was shorter (5.3 vs 7.8 min; P < 0.001) and the median ablation energy was lower (10.2 vs 18.0 kJ; P < 0.001) in multistep compared with single-step RFA. There were no significant differences in neonatal outcomes following multistep vs single-step RFA. CONCLUSIONS: Overall survival rates were similar between the two RFA methods. However, the multistep RFA technique was associated with a lower risk of fetal loss within 2 weeks after RFA. The multistep RFA technique required significantly less ablation energy and a shorter ablation time compared with single-step RFA in selective fetal reduction of MC twin and triplet pregnancies. Additionally, there was a trend towards a prolonged procedure-to-delivery interval with the multistep RFA technique. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Asperosaponin VI Protects Against Bone Loss Due to Hindlimb Unloading in Skeletally Growing Mice Through Regulating Microbial Dysbiosis Altering the 5-HT Pathway.

Osteoporosis is a complex multifactorial disease that can lead to an increased risk of fracture. However, selective and effective osteoporosis drugs are still lacking. We showed that Asperosaponin VI (AVI) has the implications to be further developed as an alternative supplement for the prevention and treatment of bone loss. AVI has been found to have beneficial effects on metabolic diseases such as bone loss, obesity, and atherosclerosis. Our study was designed to determine the effect and mechanism of action of AVI against bone loss through regulating microbial dysbiosis. A hindlimb unloading mouse model was established to determine the effect of AVI on bone microarchitecture, gut microbiota, and serum metabolites. Eighteen female C57BL/6 J mice were divided into three groups: control, hindlimb unloading with vehicle (HLU), and hindlimb unloading treated with AVI (HLU-AVI, 200 mg/kg/day). AVI was administrated orally for 4 weeks. The results demonstrated that AVI improved the bone microstructure by reversing the decrease in bone volume fraction and trabecular number, and the increase in trabecular separation and structure model index of cancellous bone in hindlimb suspension mice. The results of 16sRNA gene sequencing suggested that the therapeutic effect of AVI on bone loss may be achieved through it regulating the gut microbiota, especially certain specific microorganisms. Combined with the analysis of ELISA, immunohistochemistry, and serum metabolome results, it could be speculated that AVI played an important role in adjusting the balance of bone metabolism by influencing specific flora such as Clostridium and its metabolites to regulate the 5-hydroxytryptophan pathway. The study explored the novel mechanism of AVI against osteoporosis, and has implications for the further development of AVI as an alternative supplement for the prevention and treatment of bone loss.

Texture analysis of computed tomography data using morphologic and metabolic delineation of esophageal cancer-relation to tumor type and neoadjuvant therapy response.

The prognostic values of image-based tumor texture analysis based on computed tomography (CT) and of limiting the segmented tumor volume to metabolically active regions using fludeoxyglucose-positron emission tomography (FDG-PET) were studied in 25 patients with esophageal adenocarcinoma and 11 patients with squamous cell carcinoma. The aims of this study are to describe their CT-image-based texture characteristics before and after neoadjuvant therapy and to evaluate whether limiting the examined tumor volume to metabolically active regions detected with FDG-PET image data would further improve their value. Textural parameters (homogeneity, energy, entropy, contrast, and correlation) based on gray-level co-occurrence matrices (GLCM) were calculated for 3D volumes of segmented esophageal tumors before and after neoadjuvant chemotherapy or radiochemotherapy. Histopathological data after surgical resection and textural parameters before and after neoadjuvant treatment were compared using the Mann-Whitney U test. Significant differences in the textural parameters were observed between adenocarcinoma and squamous cell carcinoma for homogeneity, energy, inertia, and correlation. The use of contrast media during scanning resulted in significant differences in homogeneity, energy, entropy, and inertia for adenocarcinoma but not squamous cell carcinoma. There was also a significant difference in all textural parameters between pathological T status for ypT0-ypT2 and ypT3-ypT4 adenocarcinomas, but not in squamous cell carcinoma patients. No additional value was found from using PET image data to aid segmentation of CT images.

Antioxidant effect of glutathione on promoting 2-keto-l-gulonic acid production in vitamin C fermentation system.

AIMS: Oxidative stress limited the growth of cells and 2-keto-l-gulonic acid (2-KGA) production in vitamin C (Vc) fermentation system. The study aims to investigate the antioxidant effect of glutathione on promoting 2-KGA in Vc fermentation system using Ketogulonicigenium vulgare 25B-1 and Bacillus endophyticus ST-1 as the co-culturing microbes. METHODS AND RESULTS: The activities of antioxidant-related enzymes and qPCR were used to study the antioxidant effect of glutathione addition in Vc fermentation system. The addition of GSH and GSH/GSSG increased 2-KGA production and decreased fermentation time, and the highest 2-KGA production increased by 40·63% and the lowest fermentation time shortened to 60 h when the addition of optimal concentration ratio of GSH/GSSG was 50 : 1. Moreover, the increased production of 2-KGA was accompanied by up-regulated the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) and over-expressed oxidative stress-related genes sod, gst, gr, zwf, gp, which resulted in scavenging reactive oxygen species to reduce oxidative stress in Vc fermentation system. CONCLUSIONS: Glutathione showed a significant effect on increasing 2-KGA production and decreasing fermentation time in Vc fermentation system. GSH/GSSG could maintain a dynamic balance with two forms of glutathione and the optimal concentration ratio of GSH/GSSG was 50 : 1. SIGNIFICANCE AND IMPACT OF THE STUDY: Glutathione is proved to be effective to relieve oxidative stress. The promotion effects of GSSG and GSH on 2-KGA production could help to further explore the optimization of co-culture fermentation process for Vc industrial production.

Engineering a thermostable highly active glucose 6-phosphate dehydrogenase and its application to hydrogen production in vitro.

Glucose 6-phosphate dehydrogenase (G6PDH) is one of the most important dehydrogenases responsible for generating reduced NADPH for anabolism and is also the rate-limiting enzyme in the Entner-Doudoroff pathway. For in vitro biocatalysis, G6PDH must possess both high activity and good thermostability due to requirements of efficient use and low expense of biocatalyst. Here, we used directed evolution to improve thermostability of the highly active G6PDH from Zymomonas mobilis. Four generations of random mutagenesis and Petri-dish-based double-layer screening evolved the thermolabile wild-type enzyme to the thermostable mutant Mut 4-1, which showed a more than 124-fold increase in half-life time (t1/2) at 60 °C, a 3.4 °C increase in melting temperature (T m ), and a 5 °C increase in optimal temperature (Topt), without compromising the specific activity. In addition, the thermostable mutant was conducted to generate hydrogen from maltodextrin via in vitro synthetic biosystems (ivSB), gaining a more than 8-fold improvement of productivity rate with 76% of theoretical yield at 60 °C. Thus, the engineered G6PDH has been shown to effectively regenerate NADPH at high temperatures and will be applicable for NAD(P)H regeneration in numerous in vitro biocatalysis applications.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its ß-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme.

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling.

The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L(-1)⋅h(-1). The productivity was further enhanced to 54 mmol H2⋅L(-1)⋅h(-1) by increasing reaction temperature, substrate, and enzyme concentrations--an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm-2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering.

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering.

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.

Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

Association of 3q13.32 variants with hip trochanter and intertrochanter bone mineral density identified by a genome-wide association study.

We performed a GWAS of trochanter and intertrochanter bone mineral density (BMD) in the Framingham Heart Study and replicated in three independent studies. Our results identified one novel locus around the associated variations at chromosomal region 3q13.32 and replicated two loci at chromosomal regions 3p21 and 8q24. Our findings provide useful insights that enhance our understanding of bone development, osteoporosis, and fracture pathogenesis. INTRODUCTION: Hip trochanter (TRO) and intertrochanter (INT) subregions have important clinical relevance to subtrochanteric and intertrochanteric fractures but have rarely been studied by genome-wide association studies (GWASs). METHODS: Aiming to identify genomic loci associated with BMD variation at TRO and INT regions, we performed a GWAS utilizing the Framingham Heart Study (FHS, N = 6,912) as discovery sample and utilized the Women's Health Initiative (WHI) African-American subsample (N = 845), WHI Hispanic subsample (N = 446), and Omaha osteoporosis study (N = 971), for replication. RESULTS: Combining the evidence from both the discovery and the replication samples, we identified one novel locus around the associated variations at chromosomal region 3q13.32 (rs1949542, discovery p = 6.16 × 10-8, replication p = 2.86 × 10-4 for INT-BMD; discovery p = 1.35 × 10-7, replication p = 4.16 × 10-4 for TRO-BMD, closest gene RP11-384F7.1). We also replicated two loci at chromosomal regions 3p21 (rs148725943, discovery p = 6.61 × 10-7, replication p = 5.22 × 10-4 for TRO-BMD, closest gene CTNNB1) and 8q24 (rs7839059, discovery p = 2.28 × 10-7, replication p = 1.55 × 10-3 for TRO-BMD, closest gene TNFRSF11B) that were reported previously. We demonstrated that the effects at both 3q13.32 and 3p21 were specific to the TRO, but not to the femoral neck and spine. In contrast, the effect at 8q24 was common to all the sites. CONCLUSION: Our findings provide useful insights that enhance our understanding of bone development, osteoporosis, and fracture pathogenesis.

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways.

Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H2 production. Direct H2 generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H2 by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H2 generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H2 productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H2 /L h-1 , the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H2 production biosystems and artificial photosynthesis.

A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima.

Phosphopentomutase (PPM) catalyzes the interconversion of α-D-(deoxy)-ribose 1-phosphate and α-D-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on D-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl2 (0.1 mM) and 50 µM α-D-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of Km and kcat are 1.2 mM and 185 s(-1) at 90 °C, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date.

Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade.

Sugar phosphates cannot be produced easily by microbial fermentation because negatively-charged compounds cannot be secreted across intact cell membrane. D-xylulose 5-phosphate (Xu5P), a very expensive sugar phosphate, was synthesized from D-xylose and polyphosphate catalyzed by enzyme cascades in one pot. The synthetic enzymatic pathway comprised of xylose isomerase and xylulokinase was designed to produce Xu5P, along with a third enzyme, polyphosphate kinase, responsible for in site ATP regeneration. Due to the promiscuous activity of the ATP-based xylulokinase from a hyperthermophilic bacterium Thermotoga maritima on polyphosphate, the number of enzymes in the pathway was minimized to two without polyphosphate kinase. The reactions catalyzed by the two-enzyme and three-enzyme pathways were compared for Xu5P production, and the reaction conditions were optimized by examining effects of reaction temperature, enzyme ratio and substrate concentration. The optimized two-enzyme system produced 32 mM Xu5P from 50 mM xylose and polyphosphate after 36 h at 45°C. Biosynthesis of less costly Xu5P from D-xylose and polyphosphate could be highly feasible via this minimized two-enzyme pathway.

Enzymatic transformation of nonfood biomass to starch.

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose.

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to 15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.

MicroRNA-133a functions as a tumor suppressor in gastric cancer.

MicroRNAs (miRNAs) are small and highly conserved non-coding RNAs that regulate gene expression of target mRNAs through posttranscriptional inhibition involved in the tumorigenesis and progression of multiple malignancies. Although miR-133a has been shown to function as a tumor suppressor in some cancers, the clinical significance and function of miR-133a in gastric cancer remain unclear. Hence, we were focused on the expression and mechanisms of miR-133a in the development of gastric cancer in this study. It was found that the expression of miR-133a was downregulated (P<0.001), while transgelin-2 (TAGLN2) was upregulated (P<0.05) in primary gastric cancer tissues, compared to the adjacent non-cancerous tissues (ANCT). Furthermore, decreased expression of miR-133a and increased expression of TAGLN2 were both associated with lymph node metastases in patients with gastric cancer (P<0.001; P=0.011). Functional analysis studies revealed that ectopic expression of miR-133a reduced cell proliferation and invasion, and induced cell apoptosis and cycle arrest via suppressing the level of TAGLN2 from transcriptional and translational levels and downregulated the expression of proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) in gastric cancer cells. In conclusion, these results demonstrate that decreased expression of miR-133a is associated with the lymph node metastases of patients with gastric cancer. Overexpression of miR-133a inhibits cell growth and invasion and induces cell apoptosis and cycle arrest through repressing TAGLN2 gene, suggesting that miR-133a might be used as a biomarker or therapeutic target for the treatment of gastric cancer.

A new approach to determine vapour pressures and hygroscopicities of aqueous aerosols containing semi-volatile organic compounds.

We present a new approach to study the equilibrium gas-particle partitioning of volatile and semi-volatile organic components in aqueous aerosol, deriving a correlational analysis method that examines and interprets simultaneous and correlated fluctuations in particle size and composition. From this approach, changes in particle size driven by organic component evaporation can be clearly resolved from size changes driven by hygroscopicity and fluctuations in environmental conditions. The approach is used to interpret measurements of the evaporation of semi-volatile organic components from binary aqueous/organic aerosol and the hygroscopic growth of involatile inorganic aerosol. The measurements have been made by the aerosol optical tweezers technique, which allows the simultaneous retrieval of particle size and refractive index with high accuracy. We suggest that this approach will be particularly valuable for investigating the thermodynamic behaviour of mixed component aqueous aerosol and will allow the accurate derivation of solution phase equilibrium properties that are prone to large uncertainties when measurements are made simply of the change in particle size with gas phase relative humidity.

Identification and validation of a novel cuproptosis signature for stratifying different prognostic, immune, metabolic, and therapeutic landscapes in pancreatic adenocarcinoma.

OBJECTIVE: Pancreatic adenocarcinoma (PAAD) is a highly malignant cancer that urgently needs more effective therapeutic strategies. The discovery of cuproptosis brings great inspiration for the treatment and clinical assessment of cancers. MATERIALS AND METHODS: A novel cuproptosis-related (CR) risk signature was constructed using the Lasso regression analysis. Its prognostic value was assessed via a series of survival analyses and validated in four GEO cohorts. The effects of CR risk signature on tumor immune microenvironment (TIM) were explored through CIBERSORT, ESTIMATE, and ssGSEA algorithms. Using GESA, we investigated its associations with various patterns of programmed cell death (PCD) and the metabolism process. The somatic mutation features of each CR-risk group were also probed using 'maftools' R package and cBioPortal database. The potential linkages between CR risk score and the efficacy of multiple therapeutic approaches were elucidated using tumor mutation burden, the expressions of immune checkpoints, the TIDE score, and the GDSC database. Finally, we ascertained the biofunctions of LIPT1 (Lipoyltransferase 1) in pancreatic cancer (PC) cells through immunohistochemistry, qPCR (quantitative polymerase chain reaction), colony formation, and Transwell assays. RESULTS: LIPT1, LIAS (lipoyl synthase), PDP1 (Pyruvate dehydrogenase phosphatase1), and GCSH (Glycine cleavage system H protein) constituted the CR risk signature. The CR risk signature possessed a high prognostic value and could improve the traditional prognostic model. Moreover, the CR risk score was indicative of the changes in infiltration levels of CD8+T cells and macrophages, whereas it was not associated with the enrichment of various PCD patterns and multiple metabolic processes. As for therapeutic correlation, CR risk score was a potential biomarker for predicting the efficacy of ICBs but failed in targeted drugs and chemotherapeutic agents. Through qPCR and immunohistochemistry detection in clinical samples, we confirmed that LIPT1 was significantly downregulated in pancreatic adenocarcinoma (PAAD) samples. Experiments in vitro revealed that silencing LIPT1 promoted the proliferation, migration, and invasion of PANC-1 and SW1990 cells. CONCLUSIONS: The novel CR risk signature contributed to the risk stratification of PAAD patients. Cuproptosis regulatory genes, well represented by LIPT1, provided new insights into PAAD treatment and assessment.

Phylogenetic, molecular and drug-sensitivity analysis of HA and NA genes of human H3N2 influenza A viruses in Guangdong, China, 2007-2011.

Annual H3N2 subtype influenza outbreaks in Guangdong, China are a severe public health issue and require ongoing monitoring of emerging viral variants. The variation and evolution of haemagglutinin (HA) and neuraminidase (NA) genes of influenza isolates from Guangdong during 2007-2011 and others from GenBank were analysed using Lasergene 7.1 and MEGA 5.05, and serological analysis of antigens was determined by haemagglutination inhibition (HI). Susceptibility to antiviral drugs was correlated with genetic mutations. Phylogenetic analysis and alignment of HA and NA genes were performed on 18 Guangdong isolates and 26 global reference strains. The non-synonymous (dN) evolutionary rate of HA1 was 3.13 times that of HA2. Compared with the A/Perth/16/2009 vaccine HA gene, homologies of Guangdong isolates were between 98.8-99.7% and 98.0-98·4% in 2009 and 2010, respectively. Amino-acid substitutions were found in five epitopes of HA1 from Guangdong isolates between 2007 and 2011, especially in epitopes B (N160K) and D (K174R/N). The K189E/N/Q and T228A mutations in the receptor-binding site (RBS) occurred in the 2010 strains, which affected the antigenicity of HA1. The antigenicity of the epidemic H3N2 isolates in 2010 was somewhat different from that of A/Perth/16/2009. The Guangdong H3N2 isolates were determined to be oseltamivir-resistant with IC50 of 0.396 ± 0.085 nmol/l (n=17) and zanamivir-resistant with IC50 of 0.477 ± 0.149 nmol/l (n=18). Variations were present in epitopes B and D, two sites in the RBS and two glycosylation sites in the Guangdong H3N2 HA1 gene. The majority of the Guangdong H3N2 isolates were sensitive to oseltamivir and zanamivir. Compared to the World Health Organization 2012 vaccine strains, Guangdong H3N2 strains varied genetically and antigenically to some degree.