Dual specific phosphatase 4 suppresses ferroptosis and enhances sorafenib resistance in hepatocellular carcinoma.

AIMS: This investigation aims to elucidate the mechanism underlying sorafenib-induced ferroptosis in hepatocellular carcinoma (HCC). METHODS: The role of dual specificity phosphatase 4 (DUSP4) in sorafenib-treated HCC was investigated using comprehensive assessments both in vitro and in vivo, including Western blotting, qRT-PCR, cell viability assay, lipid reactive oxygen species (ROS) assay, immunohistochemistry, and xenograft tumor mouse model. Additionally, label-free quantitative proteomics was employed to identify potential proteins associated with DUSP4. RESULTS: Our study revealed that suppression of DUSP4 expression heightens the susceptibility of HCC cells to ferroptosis inducers, specifically sorafenib and erastin, in both in vitro and in vivo settings. Furthermore, we identified DUSP4-mediated regulation of key ferroptosis-related markers, such as ferritin light chain (FTL) and ferritin heavy chain 1 (FTH1). Notably, label-free quantitative proteomics unveiled the phosphorylation of threonine residue T148 on YTH Domain Containing 1 (YTHDC1) by DUSP4. Further investigations unraveled that YTHDC1, functioning as an mRNA nuclear export regulator, is a direct target of DUSP4, orchestrating the subcellular localization of FTL and FTH1 mRNAs. Significantly, our study highlights a strong correlation between elevated DUSP4 expression and sorafenib resistance in HCC. CONCLUSIONS: Our findings introduce DUSP4 as a negative regulator of sorafenib-induced ferroptosis. This discovery opens new avenues for the development of ferroptosis-based therapeutic strategies tailored for HCC treatment.

Phase separation is required for PML nuclear body biogenesis and function.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid-liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild-type (WT) NBs, PML A216V derived from arsenic-resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled-coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS-crippled and uncrippled NBs showed aggregation- and ring-like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS-driven NB formation was the prerequisite for partner recruitment, post-translational modifications (PTMs), and PML-driven cellular regulations, such as ROS stress control, mitochondria production, and PML-p53-mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.

Orexin B protects dopaminergic neurons from 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity associated with reduced extracellular signal-regulated kinase phosphorylation.

BACKGROUND: The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is a major pathological hallmark of Parkinson's disease (PD). Orexin B (OXB) has been reported to promote the growth of DA neurons. However, the roles of OXB in the degeneration of DA neurons still remained not fully clear. METHODS: An in vivo PD model was constructed by administrating 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Pole test was performed to investigate the motor function of mice and the number of DA neurons was detected by immunofluorescence (IF). A PD cell model was established by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+). OXB was added to the culture medium 2 h after MPP + treatment. Microscopic analysis was carried out to investigate the function of OXB in the cell model of PD 24 h after MPP + challenge. RNA-Seq analysis of the PD cell model was performed to explore the possible mechanisms. Western blot was used to detect the phosphorylation levels of extracellular signal-regulated kinase (ERK). RESULTS: OXB significantly decreased the DA neurons death caused by MPTP, alleviated MPP+-induced neurotoxicity in SH-SY5Y cells, and robustly enhanced the weight and motor ability of PD mice. Besides, RNA-Seq analysis demonstrated that the mitogen-activated protein kinase (MAPK) pathway was involved in the pathology of PD. Furthermore, MPP + led to increased levels of phosphorylation of ERK (p-ERK), OXB treatment significantly decreased the levels of p-ERK in MPP+-treated SH-SY5Y cells. CONCLUSIONS: This study demonstrated that OXB exerts a neuroprotective role associated with reduced ERK phosphorylation in the PD model. This suggests that OXB may have therapeutic potential for treatment of PD.

Spatial variations of fungal community assembly and soil enzyme activity in rhizosphere of zonal Stipa species in inner Mongolia grassland.

Rhizosphere soil fungal and enzyme activities affect the nutrient cycling of terrestrial ecosystems, and rhizosphere fungi are also important participants in the ecological process of vegetation succession, responding to changes in plant communities. Stipa is an excellent forage grass with important ecological and economic value, and has the spatial distribution pattern of floristic geographical substitution. In order to systematically investigate the synergistic response strategies of fungal communities and enzyme activities in the rhizosphere under the vegetation succession. Here we explored the turnover and assembly mechanisms of Stipa rhizosphere fungal communities and the spatial variation of metabolic activity under the succession of seven Stipa communities in northern China grassland under large scale gradients. The results indicated that the composition, abundance and diversity of fungal communities and microbial enzyme activities in rhizosphere soil differed among different Stipa species and were strikingly varied along the Stipa community changes over the geographic gradient. As the geographical distribution of Stipa community changed from east to west in grassland transect, Mortierellomycetes tended to be gradually replaced by Dothideomycetes. The null models showed that the rhizosphere fungal communities were governed primarily by the dispersal limitation of stochastic assembly processes, which showed decreased relative importance from S. grandis to S. gobica. Moreover, the MAT and MAP were the most important factors influencing the changes in the fungal community (richness, ß-diversity and composition) and fungal community assembly, while SC and NP also mediated fungal community assembly processes. These findings deepen our understanding of the responses of the microbial functions and fungal community assembly processes in the rhizosphere to vegetation succession.

Molecular Mechanism of Chloramphenicol and Thiamphenicol Resistance Mediated by a Novel Oxidase, CmO, in Sphingomonadaceae.

Antibiotic resistance mediated by bacterial enzyme inactivation plays a crucial role in the degradation of antibiotics in the environment. Chloramphenicol (CAP) resistance by enzymatic inactivation comprises nitro reduction, amide bond hydrolysis, and acetylation modification. However, the molecular mechanism of enzymatic oxidation of CAP remains unknown. Here, a novel oxidase gene, cmO, was identified and confirmed biochemically. The encoded CmO oxidase could catalyze the oxidation at the C-1' and C-3' positions of CAP and thiamphenicol (TAP) in Sphingobium sp. strain CAP-1. CmO is highly conserved in members of the family Sphingomonadaceae and shares the highest amino acid similarity of 41.05% with the biochemically identified glucose methanol choline (GMC) oxidoreductases. Molecular docking and site-directed mutagenesis analyses demonstrated that CAP was anchored inside the protein pocket of CmO with the hydrogen bonding of key residues glycine (G) 99, asparagine (N) 518, methionine (M) 474, and tyrosine (Y) 380. CAP sensitivity tests demonstrated that the acetyltransferase and CmO could enable a higher level of resistance to CAP than the amide bond-hydrolyzing esterase and nitroreductase. This study provides a better theoretical basis and a novel diagnostic gene for understanding and assessing the fate and resistance risk of CAP and TAP in the environment. IMPORTANCE Rising levels of antibiotic resistance are undermining ecological and human health as a result of the indiscriminate usage of antibiotics. Various resistance mechanisms have been characterized-for example, genes encoding proteins that degrade antibiotics-and yet, this requires further exploration. In this study, we report a novel gene encoding an oxidase involved in the inactivation of typical amphenicol antibiotics (chloramphenicol and thiamphenicol), and the molecular mechanism is elucidated. The findings provide novel data with which to understand the capabilities of bacteria to tackle antibiotic stress, as well as the complex function of enzymes in the contexts of antibiotic resistance development and antibiotic removal. The reported gene can be further employed as an indicator to monitor amphenicol's fate in the environment, thus benefiting risk assessment in this era of antibiotic resistance.

Complementary Biotransformation of Antimicrobial Triclocarban Obviously Mitigates Nitrous Oxide Emission toward Sustainable Microbial Denitrification.

Sustainable nitrogen cycle is an essential biogeochemical process that ensures ecosystem safety and byproduct greenhouse gas nitrous oxide reduction. Antimicrobials are always co-occurring with anthropogenic reactive nitrogen sources. However, their impacts on the ecological safety of microbial nitrogen cycle remain poorly understood. Here, a denitrifying bacterial strain Paracoccus denitrificans PD1222 was exposed to a widespread broad-spectrum antimicrobial triclocarban (TCC) at environmental concentrations. The denitrification was hindered by TCC at 25 µg L-1 and was completely inhibited once the TCC concentration exceeded 50 µg L-1. Importantly, the accumulation of N2O at 25 µg L-1 of TCC was 813 times as much as the control group without TCC, which attributed to the significantly downregulated expression of nitrous oxide reductase and the genes related to electron transfer, iron, and sulfur metabolism under TCC stress. Interestingly, combining TCC-degrading denitrifying Ochrobactrum sp. TCC-2 with strain PD1222 promoted the denitrification process and mitigated N2O emission by 2 orders of magnitude. We further consolidated the importance of complementary detoxification by introducing a TCC-hydrolyzing amidase gene tccA from strain TCC-2 into strain PD1222, which successfully protected strain PD1222 against the TCC stress. This study highlights an important link between TCC detoxification and sustainable denitrification and suggests a necessity to assess the ecological risks of antimicrobials in the context of climate change and ecosystem safety.

KLF16 enhances stress tolerance of colorectal carcinomas by modulating nucleolar homeostasis and translational reprogramming.

Translational reprogramming is part of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress, which acts to the advantage of cancer growth and development in different stress conditions, but the mechanism of ER stress-related translational reprogramming in colorectal carcinoma (CRC) progression remains unclear. Here, we identified that Krüppel-like factor 16 (KLF16) can promote CRC progression and stress tolerance through translational reprogramming. The expression of KLF16 was upregulated in CRC tissues and associated with poor prognosis for CRC patients. We found that ER stress inducers can recruit KLF16 to the nucleolus and increase its interaction with two essential proteins for nucleolar homeostasis: nucleophosmin1 (NPM1) and fibrillarin (FBL). Moreover, knockdown of KLF16 can dysregulate nucleolar homeostasis in CRC cells. Translation-reporter system and polysome profiling assays further showed that KLF16 can effectively promote cap-independent translation of ATF4, which can enhance ER-phagy and the proliferation of CRC cells. Overall, our study unveils a previously unrecognized role for KLF16 as an ER stress regulator through mediating translational reprogramming to enhance the stress tolerance of CRC cells and provides a potential therapeutic vulnerability.

Molecular Insights into the Macrophage Immunomodulatory Effects of Scrophulariae Radix Polysaccharides.

Scrophulariae Radix (SR) has been widely used in Chinese herbal compound prescriptions, health care products and functional foods. The present study aimed to investigate the immunomodulatory activity of polysaccharides from SR (SRPs) in macrophages and explore the potential mechanisms. The results showed that four SRPs fractions (SRPs40, SRPs60, SRPs80 and SRPs100) had similar absorption peaks and monosaccharide compositions, but the intensities of absorption peaks and monosaccharide contents were distinguished. All SRPs fractions significantly enhanced the pinocytic activity, promoted the production of NO and TNF-α, increased the mRNA expressions of inflammatory factors (IL-1ß, IL-6, TNF-α and PTGS2) and TLR2, and elevated the phosphorylation levels of p38, ERK, JNK, p65 and IκB. Moreover, the production of NO and TNF-α stimulated by SRPs was dramatically suppressed by anti-TLR2 antibody. These results indicated that SRPs activated macrophages through MAPK and NF-κB signaling pathways via recognition of TLR2.

Sweroside plays a role in mitigating high glucose-induced damage in human renal tubular epithelial HK-2 cells by regulating the SIRT1/NF-κB signaling pathway.

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1ß, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NF-kB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HG-caused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Complete genome sequences of the antibiotic sulfamethoxazole-mineralizing bacteria Paenarthrobacter sp. P27 and Norcardiodes sp. N27.

Sulfonamide antibiotics (SAs) have been produced and consumed on a large scale over the last few decades. SAs are a typical class of refractory contaminants that are omnipresent in various environments. Although several [phenyl]-SA-degrading bacteria and their corresponding genomes have been documented, limited genetic information is available for the degraders of heterocyclic products (e.g., 3-amino-5-methylisoxazole [3A5MI] produced via sulfamethoxazole [SMX] catabolism). In this study, the previously isolated SMX-mineralizing bacterial partners, Paenarthrobacter sp. P27 (responsible for the initial cleavage of the -C-S-N- bond of SMX and further degradation of [phenyl]-SMX) and Norcardiodes sp. N27 (responsible for 3A5MI catabolism), were further studied and their complete genomes were sequenced. Complete degradation and bacterial growth were verified by pure-culture experiments with SMX or 3A5MI as the sole carbon, nitrogen, and energy source. By cross-feeding strains P27 and N27, complete catabolism of SMX could be achieved over a wide range of initial SMX concentrations. Moreover, strain P27 was capable of transforming the additional nine SA representatives into their corresponding nitrogen-containing heterocyclic products, strongly indicating the broad substrate spectrum and marked bioremediation potential of strain P27. The genome of strain P27 contained the highly homologous monooxygenase gene cluster, sadABC, which initially attacked the sulfonamide molecules. The complete genome sequences of the two important degraders will benefit future research centering on the molecular mechanism underlying advanced SMX mineralization and will aid in further understanding the interspecific interactions and metabolite exchanges for the optimization of artificially constructed synthetic functional microbiomes.

Bioelectrochemical catabolism of triclocarban through the cascade acclimation of triclocarban-hydrolyzing and chloroanilines-oxidizing microbial communities.

Chlorinated antimicrobial triclocarban (3,4,4'-trichlorocarbanilide, TCC) is an emerging refractory contaminant omnipresent in various environments. Preferential microbial hydrolysis of TCC to chloroanilines is essential for its efficient mineralization. However, the microbial mineralization of TCC in domestic wastewater is poorly understood. Here, the bioelectrochemical catabolism of TCC to chloroanilines (3,4-dichloroaniline and 4-chloroaniline) and then to CO2 was realized through the cascade acclimation of TCC-hydrolyzing and chloroanilines-oxidizing microbial communities. The biodegradation of chloroanilines was obviously enhanced in the bioelectrochemical reactors. Pseudomonas, Diaphorobacter, and Sphingomonas were the enriched TCC or chloroanilines degraders in the bioelectrochemical reactors. The addition of TCC enhanced the synergistic effect within functional microbial communities based on the feature of the phylogenetic ecological networks. This study provides a new idea for the targeted domestication and construction of functionally differentiated microbial communities to efficiently remove TCC from domestic wastewater through a green and low-carbon bioelectrochemical method.

RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.

Retinoic acid (RA)-inducible gene I (RIG-I) is highly upregulated and functionally implicated in the RA-induced maturation of acute myeloid leukemia (AML) blasts. However, the underlying mechanism and the biological relevance of RIG-I expression to the maintenance of leukemogenic potential are poorly understood. Here, we show that RIG-I, without priming by foreign RNA, inhibits the Src-facilitated activation of AKT-mTOR in AML cells. Moreover, in a group of primary human AML blasts, RIG-I reduction renders the Src family kinases hyperactive in promoting AKT activation. Mechanistically, a PxxP motif in RIG-I, upon the N-terminal CARDs' association with the Src SH1 domain, competes with the AKT PxxP motif for recognizing the Src SH3 domain. In accordance, mutating PxxP motif prevents Rig-I from inhibiting AKT activation, cytokine-stimulated myeloid progenitor proliferation, and in vivo repopulating capacity of leukemia cells. Collectively, our data suggest an antileukemia activity of RIG-I via competitively inhibiting Src/AKT association.

ZHX3 promotes the progression of urothelial carcinoma of the bladder via repressing of RGS2 and is a novel substrate of TRIM21.

Clinically, patients with urothelial carcinoma of the bladder (UCB) with tumor metastasis are incurable. To find new therapeutic strategies, the mechanisms underlying UCB invasion and metastasis should be further investigated. In this study, zinc finger and homeobox 3 (ZHX3) was first screened as a critical oncogenic factor associated with poor prognosis in a UCB dataset from The Cancer Genome Atlas (TCGA). These results were also confirmed in a large cohort of clinical UCB clinical samples. Next, we found that ZHX3 could promote the migration and invasion capacities of UCB cells both in vitro and in vivo. Mechanistically, coimmunoprecipitation (coIP) and mass spectrometry (MS) analysis indicated that ZHX3 was a target of tripartite motif 21 (TRIM21), which mediates its ubiquitination, and subsequent degradation. Notably, RNA-seq analysis showed that ZHX3 repressed the expression of regulator of G protein signaling 2 (RGS2). Generally, our results suggest that ZHX3 plays an oncogenic role in UCB pathogenesis and might serve as a novel therapeutic target for UCB.

Purification, crystallization, and X-ray diffraction analysis of myocyte enhancer factor 2D and DNA complex.

MEF2D-fusions have recently been identified as one of the major oncogenic drivers in precursor B-cell acute lymphoblastic leukemia (B-ALL). More importantly, they are often associated with patients with poor prognosis in B-ALL. To have a better understanding of the pathogenic mechanism underpinning MEF2D-fusions-driven leukemogenesis, it's essential to uncover the related structure information. In this study, we expressed and purified the MEF2D N-terminal DNA binding domain. The recombinant protein was engineered by cloning the encoding gene into the expression vector pET-32 m. A series of chromatographic steps involving affinity, ion-exchange and gel-filtration chromatography were used to achieve a final purity of >95%. For the crystallization of the MEF2D-DNA complex, a double-stranded DNA encoding 5'-AACTATTTATAAGA-3' and 5'-TTCTTATAAATAGT-3' was used (Wu et al., 2010) [1]. The MEF2D-DNA crystal with the size of about 20 µm × 20 µm × 20 µm was obtained at a final concentration of 12 mg/ml at the reservoir condition containing 30% PEG1500. The X-ray examination showed that the MEF2D-DNA crystal diffracted to 4.5 Å resolution, and belonged to space group P1, with unit-cell parameters of a = 77.2 Å, b = 77.2 Å, c = 231.4 Å.

Microbial Interactions Drive the Complete Catabolism of the Antibiotic Sulfamethoxazole in Activated Sludge Microbiomes.

Microbial communities are believed to outperform monocultures in the complete catabolism of organic pollutants via reduced metabolic burden and increased robustness to environmental challenges; however, the interaction mechanism in functional microbiomes remains poorly understood. Here, three functionally differentiated activated sludge microbiomes (S1: complete catabolism of sulfamethoxazole (SMX); S2: complete catabolism of the phenyl part of SMX ([phenyl]-SMX) with stable accumulation of its heterocyclic product 3-amino-5-methylisoxazole (3A5MI); A: complete catabolism of 3A5MI rather than [phenyl]-SMX) were enriched. Combining time-series cultivation-independent microbial community analysis, DNA-stable isotope probing, molecular ecological network analysis, and cultivation-dependent function verification, we identified key players involved in the SMX degradation process. Paenarthrobacter and Nocardioides were primary degraders for the initial cleavage of the sulfonamide functional group (-C-S-N- bond) and 3A5MI degradation, respectively. Complete catabolism of SMX was achieved by their cross-feeding. The co-culture of Nocardioides, Acidovorax, and Sphingobium demonstrated that the nondegraders Acidovorax and Sphingobium were involved in the enhancement of 3A5MI degradation. Moreover, we unraveled the internal labor division patterns and connections among the active members centered on the two primary degraders. Overall, the proposed methodology is promisingly applicable and would help generate mechanistic, predictive, and operational understanding of the collaborative biodegradation of various contaminants. This study provides useful information for synthetic activated sludge microbiomes with optimized environmental functions.

Atomic Pt-Catalyzed Heterogeneous Anti-Markovnikov C-N Formation: Pt10 Activating N-H for Pt1δ+-Activated C═C Attack.

C-N formation is of great significance to synthetic chemistry, as N-containing products are widely used in chemistry, medicine, and biology. Addition of an amine to an unsaturated carbon-carbon bond is a simple yet effective route to produce new C-N bonds. But how to effectively conduct an anti-Markovnikov addition with high selectivity has been a great challenge. Here, we proposed a strategy for highly regioselective C-N addition via hydroamination by using supported Pt. It has been identified that atomic-scale Pt is the active site for C-N addition with Pt12+ for Markovnikov C-N formation and atomic Pt (Pt1δ+ and Pt10) contributing to anti-Markovnikov C-N formation. A selectivity of up to 92% to the anti-Markovnikov product has been achieved with atomic Pt in the addition of styrene and pyrrolidine. A cooperating catalysis for the anti-Markovnikov C-N formation between Pt1δ+ and Pt10 has been revealed. The reaction mechanism has been studied by EPR spectra and in situ FT-IR spectra of adsorption/desorption of styrene and/or pyrrolidine. It has been demonstrated that Pt10 activates amine to be electrophilic, while Pt1δ+ activates C═C by π-bonding to make ß-C nucleophilic. The attack of nucleophilic ß-C to electrophilic amine affords the anti-Markovnikov addition. This strategy proves highly effective to a variety of substrates in anti-Markovnikov C-N formation, including aromatic/aliphatic amines reacting with aromatic olefins, aromatic/aliphatic olefins with aromatic amines, and linear aliphatic olefins with secondary aliphatic amines. It is believed that the results provide evidence for the function of varied chemical states in monatomic catalysis.

How BamA recruits OMP substrates via poly-POTRAs domain.

Almost all the outer membrane proteins (OMPs) fold into an invariant ß-barrel fold via the polypeptide-transport-associated (POTRA) motif and ß-barrel assembly machinery (BAM). However, whether and how poly-POTRAs interact with OMPs remain largely unknown. Here, we have characterized the structures of Haemophilus influenzae poly-POTRAs via X-ray crystallography, small angle X-ray scattering, and molecular dynamics simulation. Unexpectedly, crystal packing reveals a putative OMP travel pathway spiraled by the conserved α2-ß2 edges in poly-POTRAs. Supportively, the structure-based mutations targeting the OMP binding sites significantly disrupt OMP biogenesis, resulting in severe cell growth defects. Another notable feature in H. influenzae POTRA structures is flexibility. As characterized by ELISA assays, poly-POTRAs could recruit OMP substrates in a step-wise manner. More importantly, the restriction of POTRA-POTRA linkage and flexibility significantly impairs the BamA function and causes cell growth defect. Altogether, these results suggest that the ß-strand augmentations and intrinsic flexibility are important factors for BamA-OMP recruitment.-Ma, X., Wang, Q., Li, Y., Tan, P., Wu, H., Wang, P., Dong, X., Hong, L., Meng, G. How BamA recruits OMP substrates via poly-POTRAs domain.

Novel Pathway for Chloramphenicol Catabolism in the Activated Sludge Bacterial Isolate Sphingobium sp. CAP-1.

The chlorinated nitroaromatic antibiotic chloramphenicol (CAP) is a refractory contaminant that is widely present in various environments. However, few CAP-mineralizing bacteria have been documented, and a complete CAP catabolism pathway has yet to be identified. In this study, the bacterial strain Sphingobium sp. CAP-1 was isolated from an activated sludge sample and was shown to be capable of aerobically subsisting on CAP as the sole carbon, nitrogen, and energy source while simultaneously and efficiently degrading CAP. p-Nitrobenzoic acid (PNBA), p-nitrobenzaldehyde (PNBD), protocatechuate (PCA), and the novel side chain C3-hydroxy-oxygenated product of CAP (O-CAP) were identified during CAP degradation. Strain CAP-1 was able to convert O-CAP to intermediate product PNBA. The putative functional genes associated with PNBA catabolism into the tricarboxylic acid cycle via PCA and floc formation were also identified by genome sequencing and comparative proteome analysis. A complete pathway for CAP catabolism was proposed. The discovery of a novel CAP oxidation/detoxification process and a complete pathway for CAP catabolism enriches the fundamental understanding of the bacterial catabolism of antibiotics, providing new insights into the microbial-mediated fate, transformation, and resistance risk of CAP in the environment. The molecular basis of CAP catabolism and floc formation in strain CAP-1 also offers theoretical guidance for the enhanced bioremediation of CAP-containing environments.

Identification of a unique 1,4-ß-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Calculation Method for Phenotypic Traits Based on the 3D Reconstruction of Maize Canopies.

A reasonable plant type is an essential factor for improving canopy structure, ensuring a reasonable expansion of the leaf area index and obtaining a high-quality spatial distribution of light. It is of great significance in promoting effective selection of the ecological breeding index and production practices for maize. In this study, a method for calculating the phenotypic traits of the maize canopy in three-dimensional (3D) space was proposed, focusing on the problems existing in traditional measurement methods in maize morphological structure research, such as their complex procedures and relatively large error margins. Specifically, the whole maize plant was first scanned with a FastSCAN hand-held scanner to obtain 3D point cloud data for maize. Subsequently, the raw point clouds were simplified by the grid method, and the effect of noise on the quality of the point clouds in maize canopies was further denoised by bilateral filtering. In the last step, the 3D structure of the maize canopy was reconstructed. In accordance with the 3D reconstruction of the maize canopy, the phenotypic traits of the maize canopy, such as plant height, stem diameter and canopy breadth, were calculated by means of a fitting sphere and a fitting cylinder. Thereafter, multiple regression analysis was carried out, focusing on the calculated data and the actual measured data to verify the accuracy of the calculation method proposed in this study. The corresponding results showed that the calculated values of plant height, stem diameter and plant width based on 3D scanning were highly correlated with the actual measured data, and the determinant coefficients R² were 0.9807, 0.8907 and 0.9562, respectively. In summary, the method proposed in this study can accurately measure the phenotypic traits of maize. Significantly, these research findings provide technical support for further research on the phenotypic traits of other crops and on variety breeding.