Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli.

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C-N bonds displays promising potential.

Microbial Dysbiosis Linked to Metabolic Dysfunction-Associated Fatty Liver Disease in Asians: Prevotella copri Promotes Lipopolysaccharide Biosynthesis and Network Instability in the Prevotella Enterotype.

Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by hepatic fat accumulation by metabolic dysfunction. The rising prevalence of MAFLD, especially among Asians, may be associated with changes in gut microbiota. We investigated gut microbiota characteristics and potential mechanisms leading to MAFLD development according to enterotypes. Case-control studies examining the gut microbiota composition between MAFLD and non-MAFLD participants were searched in public databases until July 2023. Gut microbiota was categorized into two enterotypes by principal component analysis. According to the enterotypes, LEfSe, ALDEx2, XGBoost, and DCiPatho were utilized to identify differential abundances and pathogenic microbes in the gut between the MAFLD and non-MAFLD groups. We analyzed microbial community networks with the SprCC module and predicted microbial functions. In the Prevotella enterotype (ET-P), 98.6% of Asians and 65.1% of Caucasians were associated with MAFLD (p = 0.049). MAFLD incidence was correlated with enterotype, age, obesity, and ethnicity (p < 0.05). Asian MAFLD patients exhibited decreased Firmicutes and Akkermansia muciniphila and increased Bacteroidetes and P. copri. The pathogenicity scores were 0.006 for A. muciniphila and 0.868 for P. copri. The Asian MAFLD group showed decreased stability and complexity in the gut microbiota network. Metagenome function analysis revealed higher fructose metabolism and lipopolysaccharide (LPS) biosynthesis and lower animal proteins and α-linolenic acid metabolism in Asians with MAFLD compared with the non-MAFLD group. LPS biosynthesis was positively correlated with P. copri (p < 0.05). In conclusion, P. copri emerged as a potential microbial biomarker for MAFLD. These findings enhance our understanding of the pathological mechanisms of MAFLD mediated through the gut microbiota, providing insights for future interventions.

[Chaihu Sanshen Capsules protect rats from myocardial ischemia reperfusion injury via PKCßâ ¡/NOX2/ROS signaling pathway].

This study aims to explore the pathogenesis of myocardial ischaemia reperfusion injury(MIRI) based on oxidative stress-mediated programmed cell death and the mechanism and targets of Chaihu Sanshen Capsules in treating MIRI via the protein kinase Cß(PKCßⅡ)/NADPH oxidase 2(NOX2)/reactive oxygen species(ROS) signaling pathway. The rat model of MIRI was established by the ligation of the left anterior descending branch. Rats were randomized into 6 groups: sham group, model group, clinically equivalent-, high-dose Chaihu Sanshen Capsules groups, N-acetylcysteine group, and CGP53353 group. After drug administration for 7 consecutive days, the area of myocardial infarction in each group was measured. The pathological morphology of the myocardial tissue was observed by hematoxylin-eosin(HE) staining. The apoptosis in the myocardial tissue was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). Enzyme-linked immunosorbent assay(ELISA) was employed to measure the le-vels of indicators of myocardial injury and oxidative stress. The level of ROS was detected by flow cytometry. The protein and mRNA levels of the related proteins in the myocardial tissue were determined by Western blot and real-time quantitative PCR(RT-qPCR), respectively. Compared with the sham group, the model group showed obvious myocardial infarction, myocardial structural disorders, interstitial edema and hemorrhage, presence of a large number of vacuoles, elevated levels of myocardial injury markers, myocardial apoptosis, ROS, and malondialdehyde(MDA), lowered superoxide dismutase(SOD) level, and up-regulated protein and mRNA le-vels of PKCßⅡ, NOX2, cysteinyl aspartate specific proteinase-3(caspase-3), and acyl-CoA synthetase long-chain family member 4(ACSL4) in the myocardial tissue. Compared with the model group, Chaihu Sanshen Capsules reduced the area of myocardial infarction, alleviated the pathological changes in the myocardial tissue, lowered the levels of myocardial injury and oxidative stress indicators and apoptosis, and down-regulated the mRNA and protein levels of PKCßⅡ, NOX2, caspase-3, and ACSL4 in the myocardial tissue. Chaihu Sanshen Capsules can inhibit oxidative stress and programmed cell death(apoptosis, ferroptosis) by regulating the PKCßⅡ/NOX2/ROS signaling pathway, thus mitigating myocardial ischemia reperfusion injury.

Millihertz magnetic resonance spectroscopy combining the heterodyne readout based on solid-spin sensors.

The sensitivities of quantum sensing in metrology and spectroscopy are drastically influenced by the resolution of the frequency spectrum. However, the resolution is hindered by the decoherence effect between the sensor and the environment. Along these lines, the continue-wave optically detected magnetic resonance (CWODMR) method combined with the heterodyne readout was proposed to break the limitation of the sensor's coherence time. The frequency of the magnetic field was swept to match the unknown signal, and the signal can be transformed to a real-time frequency-domain curve via the heterodyne readout, with a frequency resolution of 4.7 millihertz. Using the nitrogen-vacancy (NV) center ensemble in a diamond as the solid-spin sensors, it was demonstrated that the frequency resolution and precision could be improved proportionally to the low-pass filter parameters of Tc -1 and Tc -1.5, respectively. Furthermore, the introduced method performed the sensing of arbitrary audio signals with a sensitivity of 7.32 nT·Hz-1/2@10 kHz. Our generic approach can be extended to several fields, such as molecular structure determination and biomagnetic field detection, where high-fidelity detection properties across multiple frequency bands are required within small sensing volumes (∼ mm3).

MicroRNA alteration in cerebrospinal fluid from comatose patients with traumatic brain injury after right median nerve stimulation.

Electrical stimulation of the right median nerve can aid coma arousal after traumatic brain injury (TBI). This study aimed to confirm the efficacy further and explore possible mechanisms of right median nerve electrical stimulation (RMNS). Five comatose patients after severe TBI from May to September 2020 in the Tianjin Medical University General Hospital received RMNS for 2 weeks besides standard management. After the 2-week treatment, the mean Glasgow Coma Scale (GCS) and neurophysiological examination were used. We then investigated the alterations in microRNA (miRNA) expression in cerebrospinal fluid (CSF) by high-throughput whole transcriptome sequencing, analyzed the data by Gene Ontology (GO) and pathway analysis, and constructed the miRNA-target gene network. Patient awareness and brain function showed a more rapid increase after treatment. We also found 38 differently expressed miRNAs, 34 of which were upregulated and 4 downregulated. GO analysis showed a relation of these differentially expressed miRNAs with neuronal growth, repair, and neural signal transmission. The most highly correlated pathways were primarily associated with the tumor necrosis factor (TNF) signaling pathway and dopaminergic synapse. The application of RMNS effectively promoted early awakening in comatose patients with severe TBI. Moreover, differentially expressed miRNAs might reduce neuronal apoptosis and increase dopamine levels by regulating target gene expression, thus participating in the specific biological process after arousal therapy. Our study provided novel targets for further research on the molecular mechanisms of RMNS arousal treatment and a new way to treat neurotraumatic diseases.

Isolation and identification of mucin-degrading bacteria originated from human faeces and their potential probiotic efficacy according to host-microbiome enterotype.

AIM: Mucin-degrading bacteria are known to be beneficial for gut health. We aimed to isolate human-derived mucin-degrading bacteria and identify potential probiotic characteristics and their effects on the bacterial community and short-chain fatty acid (SCFA) production according to three different enterotypes of the host. METHODS AND RESULTS: Bacteria with mucin decomposition ability from human faeces were isolated and identified by 16S rRNA sequencing and MALDI-TOF. Heat resistance, acid resistance, antibiotic resistance, and antibacterial activity were analysed in the selected bacteria. Their adhesion capability to the Caco-2 cell was determined by scanning electron microscopy. Their ability to alter the bacterial community and SCFA production of the isolated bacteria was investigated in three enterotypes. The three isolated strains were Bifidobacterium(Bif.) animalis SPM01 (CP001606.1, 99%), Bif. longum SPM02 (NR_043437.1, 99%), and Limosilactobacillus(L.) reuteri SPM03 (CP000705.1, 99%) deposited in Korean Collection for Type Culture (KCTC-18958P). Among them, Bif. animalis exhibited the highest mucin degrading ability. They exhibited strong resistance to acidic conditions, moderate resistance to heat, and the ability to adhere tightly to Caco-2 cells. Three isolated mucin-degrading bacteria incubation increased Lactobacillus in the faecal bacteria from Bacteroides and Prevotella enterotypes. However, only L. reuteri elevated Lactobacillus in the faecal bacteria from the Ruminococcus enterotype. B. longum and B. animalis increased the α-diversity in the Ruminococcus enterotype, while their incubation with other intestinal types decreased the α-diversity. Bifidobacterium animalis and L. reuteri increased the butyric acid level in faecal bacteria from the Prevotella enterotype, and L. reuteri elevated the acetic acid level in those from the Ruminococcus enterotype. However, the overall SCFA changes were minimal. CONCLUSIONS: The isolated mucin-degrading bacteria act as probiotics and modulate gut microbiota and SCFA production differently according to the host's enterotypes. SIGNIFICANCE AND IMPACT OF STUDY: Probiotics need to be personalized according to the enterotypes in clinical application.

Temperature Fluctuations Compensation with Multi-Frequency Synchronous Manipulation for a NV Magnetometer in Fiber-Optic Scheme.

Nitrogen-vacancy (NV) centers in diamonds play a large role in advanced quantum sensing with solid-state spins for potential miniaturized and portable application scenarios. With the temperature sensitivity of NV centers, the temperature fluctuations caused by the unknown environment and the system itself will mix with the magnetic field measurement. In this research, the temperature-sensitive characteristics of different diamonds, alongside the temperature noise generated by a measurement system, were tested and analyzed with a homemade NV magnetometer in a fiber-optic scheme. In this work, a multi-frequency synchronous manipulation method for resonating with the NV centers in all axial directions was proposed to compensate for the temperature fluctuations in a fibered NV magnetic field sensing scheme. The symmetrical features of the resonance lines of the NV centers, the common-mode fluctuations including temperature fluctuations, underwent effective compensation and elimination. The fluorescence change was reduced to 1.0% by multi-frequency synchronous manipulation from 5.5% of the single-frequency manipulation within a ±2 °C temperature range. Additionally, the multi-frequency synchronous manipulation improved the fluorescence contrast and the magnetic field measurement SNR through an omnidirectional manipulation scheme. It was very important to compensate for the temperature fluctuations, caused by both internal and external factors, to make use of the NV magnetometer in fiber-optic schemes' practicality. This work will promote the rapid development and widespread applications of quantum sensing based on various systems and principles.

Beneficial Effect of Gastrodia elata Blume and Poria cocos Wolf Administration on Acute UVB Irradiation by Alleviating Inflammation through Promoting the Gut-Skin Axis.

Bioactive compounds in some herbs can, directly and indirectly, protect against photoaging. We evaluated the effects of Gastrodia elata Blume (GE) and Poria cocos Wolf (PC) water extracts on ultraviolet (UV) B-induced skin lesions by acute UVB exposure in ICR mice and explored their mechanism of action. After removing the hair on the back of the mice, UVB (280-310 nm) was exposed to the back for 30 min to induce skin damage. Four UVB exposure groups were divided into the following according to the local application (1,3-butanediol extract) on the dorsal skin and oral intake (0.3 g water extract/kg body weight/day): 1,3-butanediol and cellulose(control; UV-Con), retinoic acid (positive-control; UV-Positive), PC extracts (UV-PC), and GE extracts (UV-GE). The fifth group had no UVB exposure with the same treatment as the UV-Con (Normal-control). The erythema, burns, erosion, and wounds of the UV-PC and UV-PC groups were alleviated, and the most significant improvements occurred in the UV-PC group. PC and GE reduced the thickness of the dorsal skin tissue, the penetration of mast cells, and malondialdehyde contents. The mRNA expression of TNF-α, IL-13, and IL-4, inflammatory factors, were also reduced significantly in the dorsal skin of the UV-PC and UV-GE groups. UV-PC, UV-GE, and UV-Positive showed improvements in UV-induced intestinal tissue inflammation. UV-Con deteriorated the intestinal morphology, and PC and GE alleviated it. The α-diversity of the fecal microbiota decreased in the UV-control, and UV-PC and UV-GE prevented the decrease. Fecal metagenome analysis revealed increased propionate biosynthesis in the UV-PC group but decreased lipopolysaccharide biosynthesis in the UV-PC and UV-GE groups compared to UV-Con. In conclusion, the local application and intake of PC and GE had significant therapeutic effects on acute UV-induced skin damage by reducing oxidative stress and proinflammatory cytokines, potentially promoting the gut-microbiota-gut-skin axis.

Long-Term Effect of Porcine Brain Enzyme Hydrolysate Intake on Scopolamine-Induced Memory Impairment in Rats.

No study has revealed the effect of porcine brain enzyme hydrolysate (PBEH) on memory impairment. We aimed to examine the hypothesis that PBEH intake modulates memory deficits and cognitive behavior in scopolamine (SC)-induced amnesia rats, and its mechanism, including gut microbiota changes, was determined. Sprague-Dawley male rats had intraperitoneal injections of SC (2 mg/kg body weight/day) at 30 min after daily feeding of casein (MD-control), PBEH (7 mg total nitrogen/mL) at 0.053 mL (Low-PBEH), 0.159 mL (Medium-PBEH), 0.478 mL (High-PBEH), or 10 mg donepezil (Positive-control) per kilogram body weight per day through a feeding needle for six weeks. The Normal-control rats had casein feeding without SC injection. PBEH dose-dependently protected against memory deficits determined by passive avoidance test, Y-maze, water-maze, and novel object recognition test in SC-induced rats compared to the MD-control. The High-PBEH group had a similar memory function to the Positive-control group. Systemic insulin resistance determined by HOMA-IR was lower in the PBEH groups than in the Normal-control but not the Positive-control. In parallel with systemic insulin resistance, decreased cholesterol and increased glycogen contents in the hippocampus in the Medium-PBEH and High-PBEH represented reduced brain insulin resistance. PBEH intake prevented the increment of serum TNF-α and IL-1ß concentrations in the SC-injected rats. Hippocampal lipid peroxide and TNF-α contents and mRNA TNF-α and IL-1ß expression were dose-dependently reduced in PBEH and Positive-control. PBEH decreased the hippocampal acetylcholinesterase activity compared to the MD-control, but not as much as the Positive-control. PBEH intake increased the α-diversity of the gut microbiota compared to the MD-control, and the gut microbiota community was separated from MD-control. In metagenome function analysis, PBEH increased the energy metabolism-related pathways of the gut microbiota, including citric acid cycle, oxidative phosphorylation, glycolysis, and amino acid metabolism, which were lower in the MD-control than the Normal-control. In conclusion, alleviated memory deficit by PBEH was associated potentially with not only reducing acetylcholinesterase activity but also improving brain insulin resistance and neuroinflammation potentially through modulating gut microbiota. PBEH intake (1.5-4.5 mL of 7 mg total nitrogen/mL for human equivalent) can be a potential therapeutic agent for improving memory impairment.

A noninvasive index to predict liver cirrhosis in biliary atresia.

BACKGROUND: Biliary atresia is a progressive obliterative cholangiopathy affecting both extrahepatic and intrahepatic biliary trees, resulting in fibrous obliteration of the biliary tract and subsequent development of cirrhosis. OBJECTIVE: The aim of this study was to find noninvasive indices to predict the status of hepatic fibrosis in children with biliary atresia. MATERIALS AND METHODS: We retrospectively measured the volume of the hepatic lobes and spleen from MR images, obtained biochemical data and analyzed the relationship between the imaging and biochemical indices, and the pathological status of hepatic fibrosis in 35 children with biliary atresia. RESULTS: A combined index was obtained by logistic regression: logit (likelihood of cirrhosis) = 0.00043 x age at MR examination + 1.67 x aspartate aminotransferase and platelet ratio index (APRI) + 0.0029 x body-surface-area-adjusted left liver lobe volume (BSA adLLV) - 6.57 (log-likelihood chi-square P<0.05, pseudo-R2=0.59). The area under the receiver operator characteristic curve of age at MR examination, APRI, BSA adLLV and the combined index for prediction of cirrhosis were 0.91, 0.86, 0.83 and 0.94, respectively. The optimal cut-off value (sensitivity and specificity) of age at MR examination, APRI, BSA adLLV and combined index were 132 (86% and 92%), 1.3 (91% and 85%), 855.5 (96% and 62%) and 0.689 (91% and 92%). The accuracy of age at MR examination, APRI, BSA adLLV and combined index were 89%, 89%, 83% and 91%, respectively. CONCLUSION: A combined noninvasive index of age, aspartate aminotransferase and platelet ratio index, and the body-surface-area-adjusted left liver lobe volume measured from MR images is a potential marker of liver cirrhosis in children with biliary atresia.

Epidemiological analysis of 67 local COVID-19 clusters in Sichuan Province, China.

BACKGROUND: This study was intended to investigate the epidemiological characteristics of COVID-19 clusters and the severity distribution of clinical symptoms of involved cases in Sichuan Province, so as to provide information support for the development and adjustment of strategies for the prevention and control of local clusters. METHODS: The epidemiological characteristics of 67 local clusters of COVID-19 cases in Sichuan Province reported as of March 17, 2020 were described and analyzed. Information about all COVID-19 clusters and involved cases was acquired from the China Information System for Disease Control and Prevention and analyzed with the epidemiological investigation results taken into account. RESULTS: The clusters were temporally and regionally concentrated. Clusters caused by imported cases from other provinces accounted for 73.13%; familial clusters accounted for 68.66%; the average attack rate was 8.54%, and the average secondary attack rate was 6.11%; the median incubation period was 8.5 d; a total of 28 cases met the criteria for incubation period determination, and in the 28 cases, the incubation period was > 14 d in 21.43% (6/28). a total of 226 confirmed cases were reported in the 67 clusters. Ten cases were exposed before the confirmed cases they contacted with developed clinical symptoms, and the possibility of exposure to other infection sources was ruled out; two clusters were caused by asymptomatic carriers; confirmed cases mainly presented with fever, respiratory and systemic symptoms; a gradual decline in the severity of clinical symptoms was noted with the increase of the case generation. CONCLUSIONS: Population movement and gathering restrictions and strict close contact management measures will significantly contribute to the identification and control of cases. Transmission during the incubation period and asymptomatic infections have been noted. Studies on the pathogenicity and transmissibility in these populations and on COVID-19 antibody levels and protective effects in healthy people and cases are required.

High-efficiency fluorescence collection for NV- center ensembles in diamond.

The negatively charged nitrogen vacancy (NV-) center ensembles in diamond have been demonstrated to be a promising platform for quantum metrology, but the poor fluorescence collection efficiency of a microscope objective limits the sensitivity of the NV- based sensors. Here we present a method for increasing the collected fluorescence intensity with a total internal reflection (TIR) lens. The detected fluorescence intensity is increased by approximately a factor of 56 compared with detection using a microscope objective with NA = 0.55, leading to a collection efficiency of 47.7% ± 3.1%. The signal-to-noise ratio is improved by a factor of 7.6 using the TIR lens. The proposed method is of great significance for collecting fluorescence from NV- centers in a large volume and can be used in weak fluorescence detection systems.

Antitumor effect of axitinib combined with dopamine and PK-PD modeling in the treatment of human breast cancer xenograft.

Rising evidence has shown the development of resistance to vascular endothelial growth factor receptor (VEGFR) inhibitors in the practices of cancer therapy. It is reported that the efficacy of axitinib (AX), a VEGFR inhibitor, is limited in the treatment of breast cancer as a single agent or in combination with other chemotherapeutic drugs due to the probability of rising population of cancer stem-like cells (CSCs) caused by AX. The present study evaluated the effect of dopamine (DA) improving AX's efficacy on MCF-7/ADR breast cancer in vitro and in vivo, and developed a pharmacokinetic-pharmacodynamic (PK-PD) model describing the in vivo experimental data and characterizing the interaction of effect between AX and DA. The results showed that AX up-regulated the expression of breast CSC (BCSC) markers (CD44+/CD24-/low) in vivo, and DA significantly synergized the inhibitory effect on tumor growth by deducting the BCSC frequency. The PK-PD model quantitatively confirmed the synergistic interaction with the parameter estimate of interaction factor ψ 2.43. The dose regimen was optimized as 60 mg/kg AX i.g. b.i.d. combined with 50 mg/kg DA i.p. q3d in the simulation study on the basis of the PK-PD model. The model where DA synergistically enhances the effect of AX in an all-or-none manner provides a possible solution in modeling the agents like DA. Moreover, the outcome of AX and DA combination therapy in MCF-7/ADR breast cancer provided further insight of co-administering DA in the treatment of the possible CSC-causing AX-resisting breast cancer. And this combination therapy has the prospect of clinical translation.

Inhibition of G9a by a small molecule inhibitor, UNC0642, induces apoptosis of human bladder cancer cells.

Urinary bladder cancer (UBC) is characterized by frequent recurrence and metastasis despite the standard chemotherapy with gemcitabine and cisplatin combination. Histone modifiers are often dysregulated in cancer development, thus they can serve as an excellent drug targets for cancer therapy. Here, we investigated whether G9a, one of the histone H3 methyltransferases, was associated with UBC development. We first analyzed clinical data from public databases and found that G9a was significantly overexpressed in UBC patients. The TCGA Provisional dataset showed that the average expression level of G9a in primary UBC samples (n = 408) was 1.6-fold as much as that in normal bladder samples (n = 19; P < 0.001). Then we used small interfering RNA to knockdown G9a in human UBC T24 and J82 cell lines in vitro, and observed that the cell viability was significantly decreased and cell apoptosis induced. Next, we choosed UNC0642, a small molecule inhibitor targeting G9a, with low cytotoxicity, and excellent in vivo pharmacokinetic properties, to test its anticancer effects against UBC cells in vitro and in vivo. Treatment with UNC0642 dose-dependently decreased the viability of T24, J82, and 5637 cells with the IC50 values of 9.85 ± 0.41, 13.15 ± 1.72, and 9.57 ± 0.37 µM, respectively. Furthermore, treatment with UNC0642 (1-20 µM) dose-dependently decreased the levels of histone H3K9me2, the downstream target of G9a, and increased apoptosis in T24 and J82 cells. In nude mice bearing J82 engrafts, administration of UNC0642 (5 mg/kg, every other day, i.p., for 6 times) exerted significant suppressive effect on tumor growth without loss of mouse body weight. Moreover, administration of UNC0642 significantly reduced Ki67 expression and increased the level of cleaved Caspase 3 and BIM protein in J82 xenografts evidenced by immunohistochemistry and western blot analysis, respectively. Taken together, our data demonstrated that G9a may be a promising therapeutic target for UBC, and an epigenetics-based therapy by UNC0642 is suggested.

Structural effects of extracellular loop mutations in CFTR helical hairpins.

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.

Compact birefringent interferometer for Fourier transform hyperspectral imaging.

A compact birefringent interferometer (CBI) for Fourier transform hyperspectral imaging is presented. The CBI employs only two birefringent crystal plates: a shearing plate (SP) and a compensation plate (CP). The SP generates the optical path difference (OPD) associated with the field of view for broadband interference. The CP compensates the constant term and square term OPDs of the SP to adjust the position of the zero-order fringe pattern and suppress inconsistent total OPDs and other nonlinear OPDs. This paper details the theoretically deduced OPDs and then presents simulation analyses and verification experiments conducted to investigate the OPD distribution characteristics. To verify the CBI performance, experimental spectral measurements and hyperspectral imaging were performed. The experimental results demonstrate that the CBI can suppress inconsistent total OPDs and other nonlinear OPDs with only two birefringent crystal plates, and therefore offers much promise for miniature and high-precision Fourier transform hyperspectral imaging.

Ultra-compact Fourier transform imaging spectrometer using a focal plane birefringent interferometer.

A new Fourier transform imaging spectrometer based on a focal plane birefringent interferometer (FPBI) is presented. The FPBI, located in front of the detector, is capable of performing spectral imaging measurements. It mainly consists of a birefringent plate and a birefringent wedge. The ordinary and extraordinary rays-with an optical path difference-are split by the FPBI and interfere on the focal plane. The spectral image of the target can be acquired via scene scanning and spectral recovery. The principle of interferometric imaging of the FPBI is investigated, and verification experiments are then performed. The experiments indicate that the FPBI not only provides effective spectral imaging measurements, but also presents the advantages of being ultra-compact and lightweight. As a result, it can be effectively applied in situations such as outdoor surveillance and airborne remote sensing.

Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer.

BACKGROUND: Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients. METHODS: First, acquired cetuximab-resistant cell lines were generated, and UCA1 expressions in these cells and their exosomes were compared. We also systematically evaluate the stability of exosomal UCA1. Thereafter, the predictive value of exosomal UCA1 in CRC patients treated with cetuximab was evaluated. Finally, through cell apoptosis assays and immunofluorescence staining, we analyzed the role of UCA1-containing exosomes in conferring cetuximab resistance. RESULTS: UCA1 expression was markedly higher in cetuximab-resistant cancer cells and their exosomes. Exosomal UCA1 was shown to be detectable and stable in serum from CRC patients. In addition, circulating UCA1-containing exosomes could predict the clinical outcome of cetuximab therapy in CRC patients, and UCA1 expression was considerably higher in the progressive disease/stable disease patients than in the partial response/complete response patients. Furthermore, exosomes derived from cetuximab-resistant cells could alter UCA1 expression and transmit cetuximab resistance to sensitive cells. CONCLUSIONS: We discovered a novel role of UCA1-containing exosomes, showed their capability to transmit drug resistance and investigated their potential clinical use in predicting cetuximab resistance.

Biochemical Characterization and Degradation Pattern of a Unique pH-Stable PolyM-Specific Alginate Lyase from Newly Isolated Serratia marcescens NJ-07.

Enzymatic preparation of alginate oligosaccharides with versatile bioactivities by alginate lyases has attracted increasing attention due to its featured characteristics, such as wild condition and specific products. In this study, AlgNJ-07, a novel polyM-specific alginate lyase with high specific activity and pH stability, has been purified from the newly isolated marine bacterium Serratia marcescens NJ-07. It has a molecular weight of approximately 25 kDa and exhibits the maximal activity of 2742.5 U/mg towards sodium alginate under 40 °C at pH 9.0. Additionally, AlgNJ-07 could retain more than 95% of its activity at pH range of 8.0-10.0, indicating it possesses excellent pH-stability. Moreover, it shows high activity and affinity towards polyM block and no activity to polyG block, which suggests that it is a strict polyM-specific alginate lyase. The degradation pattern of AlgNJ-07 has also been explored. The activity of AlgNJ-07 could be activated by NaCl with a low concentration (100-300 mM). It can be observed that AlgNJ-07 can recognize the trisaccharide as the minimal substrate and hydrolyze the trisaccharide into monosaccharide and disaccharide. The TLC and ESI-MS analysis indicate that it can hydrolyze substrates in a unique endolytic manner, producing not only oligosaccharides with Dp of 2-5 but also a large fraction of monosaccharide. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower Dps (degree of polymerization).

PDTC ameliorates decompression induced-lung injury caused by unsafe fast buoyancy ascent escape via inhibition of NF-κB pathway.

Nuclear factor kappa B (NF-κB) is the critical transcriptional factor in the pathogenesis of acute lung injury (ALI). NF-κB regulates the expression changes of inflammatory factors such as tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin 6 (IL-6). In a previous study we showed that decompression sickness (DCS) caused by simulated unsafe fast buoyancy ascent escape (FBAE) could result in ALI, which was characterized by expression changes of inflammatory factors in rat lung tissue. The purpose of the present work was to study the roles of NF-κB and TNF-α in the process of DCS-induced rat lung injury caused by simulated unsafe FBAE. The research methods aimed to detect the rat lung tissue messenger ribonucleic acid (mRNA) and protein level variations of NF-κB, inhibitory ×B (I×B), TNF-α, IL-1ß, IL-6, IL-10 and IL-13 by using pretreatment of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and TNF-α antibody (Ab). Our experimental results demonstrated that PDTC could improve the survival rate of the rats with DCS caused by unsafe FBAE more effectively than TNF-α Ab. However, the inhibition of TNF-α Ab on the nuclear translocated protein expression of NF-κB was more effective than PDTC. Both PDTC and TNF-α Ab can abrogate the increment of the rat lung tissue mRNA levels of TNF-α, IL-1ß, IL-6 and protein levels of NF-κB, TNF-α, IL-1ß effectively and increase the rat lung tissue content of I×B significantly. In conclusion, TNF-α-mediated NF-κB signaling may be one of the critical signaling pathways in the pathogenesis of DCS-induced rat lung injury caused by simulated unsafe FBAE. PDTC may ameliorate this type of injury partly through inhibiting the NF-κB pathway.