Plasma neutrophil gelatinase-associated lipocalin predicts renal tubulointerstitial injury in patients with IgA nephropathy.

OBJECTIVE: To investigate the relationship of plasma neutrophil gelatinase-associated lipocalin (pNGAL) and urine neutrophil gelatinase-associated lipocalin (uNGAL) with clinical indicators and pathology in patients with IgA nephropathy (IgAN). MATERIALS AND METHODS: Clinical and pathological data of 36 patients with primary IgAN diagnosed by kidney biopsy were selected. The Oxford and Lee classifications were used to rank the pathology of IgAN patients and the particle-enhanced immunoturbidimetric method was adopted to measure pNGAL and uNGAL. Subsequently, the correlations of pNGAL and uNGAL levels with clinical indices and pathology were analyzed to evaluate the diagnostic value. RESULTS: Referring to the comparison group, pNGAL levels were elevated in the women, 24-hour proteinuria ≥ 1 g/day, Lee classification IV - V, and tubular atrophy/interstitial fibrosis (T) score of 1 subgroup of Oxford classification (p < 0.05). The pNGAL was positively correlated with 24-hour proteinuria, urine microalbumin, urine α1 microglobulin, and the score of T (p < 0.05), while uNGAL was negatively correlated with serum albumin (p < 0.05). The occurrence of T1 was evaluated by pNGAL, serum creatinine (Scr), 24-hour proteinuria, and estimated glomerular filtration rate (eGFR), and it was shown that the area under the curve of 24-hour proteinuria, pNGAL, Scr, and eGFR were 0.629, 0.817, 0.919, and 0.799, respectively; in addition, the sensitivity of both pNGAL and eGFR are 100%. CONCLUSION: The pNGAL levels in patients with IgAN are positively correlated with the Lee and Oxford classifications of T scores. The level of pNGAL in IgAN patients is found to be a prominent indicator for identification of renal tubulointerstitial injury and perhaps this feature may make it a non-invasive predictor for progression of IgAN.

Diagnostic value of 3D volume measurement of central pulmonary artery based on CTPA images in the pulmonary hypertension.

BACKGROUND: This retrospective study aims to evaluate the diagnostic value of volume measurement of central pulmonary arteries using computer tomography pulmonary angiography (CTPA) for predicting pulmonary hypertension (PH). METHODS: A total of 59 patients in our hospital from November 2013 to April 2021 who underwent both right cardiac catheterization (RHC) and CTPA examination were included. Systolic pulmonary artery pressure (SPAP), mean PAP (mPAP), and diastolic PAP (DPAP) were acquired from RHC testing. Patients were divided into the non-PH group (18 cases) and the PH group (41 cases). The diameters of the main pulmonary artery (DMPA), right pulmonary artery (DRPA), and left pulmonary artery (DLPA) were measured manually. A 3D model software was used for the segmentation of central pulmonary arteries. The cross-sectional areas (AMPA, ARPA, ALPA) and the volumes (VMPA, VRPA, VLPA) were calculated. Measurements of the pulmonary arteries derived from CTPA images were compared between the two groups, and correlated with the parameters of RHC testing. ROC curves and decision curve analysis (DCA) were used to evaluate the benefit of the three-dimensional CTPA parameters for predicting PH. A multiple linear regression model with a forward-step approach was adopted to integrate all statistically significant CTPA parameters for PH prediction. RESULTS: All parameters (DMPA, DRPA, DLPA, AMPA, ARPA, ALPA, VMPA, VRPA, and VLPA) of CTPA images exhibited significantly elevated in the PH group in contrast to the non-PH group (P < 0.05), and showed positive correlations with the parameters of RHC testing (mPAP, DPAP, SPAP) (r ranged 0.586~0.752 for MPA, 0.527~0.640 for RPA, and 0.302~0.495 for LPA, all with P < 0.05). For the MPA and RPA, 3D parameters showed higher correlation coefficients compared to their one-dimensional and two-dimensional counterparts. The ROC analysis indicated that the VMPA showed higher area under the curves (AUC) than the DMPA and AMPA without significance, and the VRPA showed higher AUC than the DRPA and ARPA significantly (DRPA vs. VRPA, Z = 2.029, P = 0.042; ARPA vs. VRPA, Z = 2.119, P = 0.034). The DCA demonstrated that the three-dimensional parameters could provide great net benefit for MPA and RPA. The predictive equations for mPAP, DPAP, and SPAP were formulated as [8.178 + 0.0006 * VMPA], [1.418 + 0.0005 * VMPA], and [-11.137 + 0.0006*VRPA + 1.259 * DMPA], respectively. CONCLUSION: The 3D volume measurement of the MPA and RPA based on CTPA images maybe more informative than the traditional diameter and cross-sectional area in predicting PH.

Metabolic engineering of genome-streamlined strain Pseudomonas putida KTU-U27 for medium-chain-length polyhydroxyalkanoate production from xylose and cellobiose.

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-Ptac-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.

Serum Uric Acid Levels Are Related to Diabetic Peripheral Neuropathy, Especially for Motor Conduction Velocity of Tibial Nerve in Type 2 Diabetes Mellitus Patients.

Background: Oxidative stress is one of the most critical factors that contribute to the pathogenesis of neuronal damage, including diabetic peripheral neuropathy (DPN). Uric acid is a kind of natural antioxidant that plays a major role in the antioxidant capacity against oxidative stress. Here, we aim to determine the role of serum uric acid (SUA) in the DPN of patients with type 2 diabetes mellitus (T2DM). Patients and Methods. 106 patients with T2DM were recruited and divided into the DPN group and the control group. Clinical parameters, especially for motor nerve fiber conduction velocity and sensory nerve fiber conduction velocity, were collected. Differences between T2DM patients with and without DPN were compared. Correlation and regression analyses were performed to explore the association between SUA and DPN. Results: Compare with 57 patients with DPN, 49 patients without DPN showed lower HbA1c and elevated SUA levels. Additionally, SUA levels are negatively associated with the motor conduction velocity of tibial nerve with or without adjusting for HbA1c. Besides, it is suggested that decreased SUA levels may influence the motor conduction speed of the tibial nerve by multiple linear regression analysis. Moreover, we demonstrated that decreased SUA level is a risk factor for DPN in patients with T2DM by binary logistic regression analysis. Conclusion: Lower SUA is a risk factor for DPN in patients with T2DM. Additionally, decreased SUA may influence the damage of peripheral neuropathy, especially for motor conduction velocity of the tibial nerve.

QMRA of beach water by Nanopore sequencing-based viability-metagenomics absolute quantification.

The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.

Creating an efficient 1,2-dichloroethane-mineralizing bacterium by a combination of pathway engineering and promoter engineering.

Currently, 1,2-dichloroethane (DCA) is frequently detected in groundwater and has been listed as a potential human carcinogen by the U.S. EPA. Owing to its toxicity and recalcitrant nature, inefficient DCA mineralization has become a bottleneck of DCA bioremediation. In this study, the first engineered DCA-mineralizing strain KTU-P8DCA was constructed by functional assembly of DCA degradation pathway and enhancing pathway expression with a strong promoter P8 in the biosafety strain Pseudomonas putida KT2440. Strain KTU-P8DCA can metabolize DCA to produce CO2 and utilize DCA as the sole carbon source for cell growth by quantifying 13C stable isotope ratios in collected CO2 and in lyophilized cells. Strain KTU-P8DCA exhibited superior tolerance to high concentrations of DCA. Excellent genetic stability was also observed in continuous passage culture. Therefore, strain KTU-P8DCA has enormous potential for use in bioremediation of sites heavily contaminated with DCA. In the future, our strategy for pathway construction and optimization is expected to be developed as a standard pipeline for creating a wide variety of new contaminants-mineralizing microorganisms. The present study also highlights the power of synthetic biology in creating novel degraders for environmental remediation.

Unsterile production of a polyhydroxyalkanoate copolymer by Halomonas cupida J9.

Microbial production of bioplastics polyhydroxyalkanoates (PHA) has opened new avenues to resolve "white pollution" caused by petroleum-based plastics. PHAs consisting of short- and medium-chain-length monomers, designated as SCL-co-MCL PHAs, exhibit much better thermal and mechanical properties than PHA homopolymers. In this study, a halophilic bacterium Halomonas cupida J9 was isolated from highly saline wastewater and proven to produce SCL-co-MCL PHA consisting of 3-hydroxybutyrate (3HB) and 3-hydroxydodecanoate (3HDD) from glucose and glycerol. Whole-genome sequencing and functional annotation suggest that H. cupida J9 may possess three putative PHA biosynthesis pathways and a class I PHA synthase (PhaCJ9). Interestingly, the purified His6-tagged PhaCJ9 from E. coli BL21 (DE3) showed polymerizing activity towards 3HDD-CoA and a phaCJ9-deficient mutant was unable to produce PHA, which indicated that a low-substrate-specificity PhaCJ9 was exclusively responsible for PHA polymerization in H. cupida J9. Docking simulation demonstrated higher binding affinity between 3HB-CoA and PhaCJ9 and identified the key residues involved in hydrogen bonds formation between 3-hydroxyacyl-CoA and PhaCJ9. Furthermore, His489 was identified by site-specific mutagenesis as the key residue for the interaction of 3HDD-CoA with PhaCJ9. Finally, PHA was produced by H. cupida J9 from glucose and glycerol in shake flasks and a 5-L fermentor under unsterile conditions. The open fermentation mode makes this strain a promising candidate for low-cost production of SCL-co-MCL PHAs. Especially, the low-specificity PhaCJ9 has great potential to be engineered for an enlarged substrate range to synthesize tailor-made novel SCL-co-MCL PHAs.

Enhanced production of polyhydroxyalkanoates in Pseudomonas putida KT2440 by a combination of genome streamlining and promoter engineering.

Polyhydroxyalkanoates (PHAs), a class of bioplastics produced by a variety of microorganisms, have become the ideal alternatives for oil-derived plastics due to their superior physicochemical and material characteristics. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from various substrates. In this study, a novel strategy of the large-scale deletion of genomic islands (GIs) coupling with promoter engineering was developed in P. putida KT2440 for constructing the minimal genome cell factories (MGF) capable of efficiently producing mcl-PHA. Firstly, P. putida KTU-U13, a 13 GIs- and upp-deleted mutant derived from the parental strain P. putida KT2440, was used as a starting strain for further deletion of GIs to generate a series of genome-reduced strains. Subsequently, the two minimal genome strains KTU-U24 and KTU-U27, which had a 7.19% and 8.35% reduction relative to the genome size of KT2440 and were advantageous over the strain KTU (KT2440∆upp) and KTU-U13 in several physiological traits such as the maximum specific growth rate, plasmid transformation efficiency, heterologous protein expression capacity and PHA production capacity, were selected as the chassis cells for PHA metabolic engineering. To prevent the formation of the by-product gluconic acid, the glucose dehydrogenase gene was deleted in KTU-U24 and KTU-U27, resulting in KTU-U24∆gcd and KTU-U27∆gcd. To enhance the transcriptional level of PHA synthase genes (phaC) and the supply of the precursor acetyl-CoA, a strong endogenous promoter P46 was inserted into upstream of the phaC operon and pyruvate dehydrogenase gene in the genome of KTU-U24∆gcd and KTU-U27∆gcd, to generate KTU-U24∆gcd-P46CA and KTU-U27∆gcd-P46CA, with the PHA yield of 50.5 wt% and 53.8 wt% (weight percent of PHA in cell dry weight). Finally, KTU-U27∆gcd-P46CA, the most minimal KT2440 chassis currently available, was able to accumulate the PHA to 55.82 wt% in a 5-l fermentor, which is the highest PHA yield obtained with P. putida KT2440 so far. This study suggests that genome streamlining in combination with promoter engineering may be a feasible strategy for the development of the MGF for the efficient production of high value products. Moreover, further streamlining of the P. putida KT2440 genome has great potential to create the optimal chassis for synthetic biology applications.

AdpA, a developmental regulator, promotes ε-poly-L-lysine biosynthesis in Streptomyces albulus.

BACKGROUND: AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces. RESULTS: In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus. CONCLUSIONS: Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Synergistic Intra- and Intermolecular Noncovalent Interactions for Ultralong Organic Phosphorescence.

Metal-free ultralong organic phosphorescence (UOP) materials have attracted significant attention owing to their anomalous photophysical properties and potential applications in various fields. Here, three pyrimidine-based organic luminogens, 9-(pyrimidin-2-yl)-9H-carbazole, 9-(4,6-dimethylpyrimidin-2-yl)-9H-carbazole, and 9-(5-bromopyrimidin-2-yl)-9H-carbazole are designed and synthesized, which show efficient yellow UOP with the longest lifetimes up to 1.37 s and the highest absolute phosphorescence quantum yields up to 23.6% under ambient conditions. Theoretical calculations, crystal structures, and photophysical properties of these compounds reveal that intramolecular hydrogen bonding, intermolecular π-π interactions, and intermolecular electronic coupling are responsible for forming dimers and generating highly efficient UOP. Their efficacy as solid materials for data encryption is demonstrated.