ABSTRACT
Collective excitations including plasmons, magnons, and layer-breathing vibration modes emerge at an ultralow frequency (<1 THz) and are crucial for understanding van der Waals materials. Strain at the nanoscale can drastically change the property of van der Waals materials and create localized states like quantum emitters. However, it remains unclear how nanoscale strain changes collective excitations. Herein, ultralow-frequency tip-enhanced Raman spectroscopy (TERS) with sub-10 nm resolution under ambient conditions is developed to explore the localized collective excitation on monolayer semiconductors with nanoscale strains. A new vibrational mode is discovered at around 12 cm-1 (0.36 THz) on monolayer MoSe2 nanobubbles and it is identified as the radial breathing mode (RBM) of the curved monolayer. The correlation is determined between the RBM frequency and the strain by simultaneously performing deterministic nanoindentation and TERS measurement on monolayer MoSe2. The generality of the RBM in nanoscale curved monolayer WSe2 and bilayer MoSe2 is demonstrated. Using the RBM frequency, the strain of the monolayer MoSe2 on the nanoscale can be mapped. Such an ultralow-frequency vibration from curved van der Waals materials provides a new approach to study nanoscale strains and points to more localized collective excitations to be discovered at the nanoscale.
ABSTRACT
There is growing interest in developing a high-performance self-supervised denoising algorithm for real-time chemical hyperspectral imaging. With a good understanding of the working function of the zero-shot Noise2Noise-based denoising algorithm, we developed a self-supervised Signal2Signal (S2S) algorithm for real-time denoising with a single chemical hyperspectral image. Owing to the accurate distinction and capture of the weak signal from the random fluctuating noise, S2S displays excellent denoising performance, even for the hyperspectral image with a spectral signal-to-noise ratio (SNR) as low as 1.12. Under this condition, both the image clarity and the spatial resolution could be significantly improved and present an almost identical pattern with a spectral SNR of 7.87. The feasibility of real-time denoising during imaging was well demonstrated, and S2S was applied to monitor the photoinduced exfoliation of transition metal dichalcogenide, which is hard to accomplish by confocal Raman spectroscopy. In general, the real-time denoising capability of S2S offers an easy way toward in situ/in vivo/operando research with much improved spatial and temporal resolution. S2S is open-source at https://github.com/3331822w/Signal2signal and will be accessible online at https://ramancloud.xmu.edu.cn/tutorial.
ABSTRACT
Necrotizing fasciitis is a rare, severe, rapidly progressing disease with a high mortality rate. We report a case of a 72-year female with erythematous pemphigus who developed erythema, swelling and ulceration on right vulva, groin, and thigh. The early clinical manifestations of the patient were nonspecific and easily misdiagnosed as cellulitis. However, upon the occurrence of ulceration and necrosis, deep fungal infection, pyoderma gangrenosum or lymphoproliferative disorders were considered. The pathology suggested IgG4-related diseases, plasmacytoma et al. But at last, surgical exploration and postoperative pathology confirmed the diagnosis of necrotizing fasciitis. The patient recovered after multiple aggressive surgical debridement procedures and antibiotic therapy and the patient has been followed up for 2 years without recurrence. Clinicians should be vigilant about the possibility of necrotizing fasciitis in patients with erythema, pain, rapid ulceration of skin and soft tissue, particularly in immunocompromised individuals with long-term use of immunosuppressive agents. It is crucial for saving life by early multi-disciplinary consultation, prompt diagnosis, and aggressive treatment.
ABSTRACT
Despite great advances in protein structure analysis, label-free and ultrasensitive methods to obtain the natural and dynamic three-dimensional (3D) structures are still urgently needed. Surface-enhanced Raman spectroscopy (SERS) can be a good candidate, whereas the complexity originated from the interactions between the protein and the gradient surface electric field makes it extremely challenging to determine the protein structure. Here, we propose a deciphering strategy for accurate determination of 3D protein structure from experimental SERS spectra in seconds by simply summing SERS spectra of isolated amino acids in electric fields of different strength with their orientations in protein. The 3D protein structure can be reconstructed by comparing the experimental spectra obtained in a well-defined gap-mode SERS configuration with the simulated spectra. The gradient electric field endows SERS with a unique advantage to section biomolecules with atomic precision, which makes SERS a competent tool for monitoring biomolecular events under physiological conditions.
Subject(s)
Proteins , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Amino AcidsABSTRACT
Recovering phosphorus from incineration sludge sewage ash (ISSA) is a well-established technology, with a greater recovery potential than that of supernatant or sludge. ISSA can be utilized as a secondary raw material in the fertilizer industry, or as a fertilizer if heavy metal concentrations do not exceed permissible limits, thus reducing the cost of phosphorus recovery. Increasing the temperature to produce ISSA with higher solubility and plant availability of phosphorus is advantageous for both pathways. But a decrease in the extraction of phosphorus is also observed at high temperatures, thereby diminishing the overall economic benefits. In this study, CaCl2 was utilized to mitigate the decrease in the extraction rate and also to promote the bioavailability of phosphorus. The addition of CaCl2 (80 g/kg of dry sludge) effectively promoted the conversion of non-apatite inorganic phosphorus to apatite inorganic phosphorus at a rate of 87.73% at 750 °C. Furthermore, the decrease in the extraction rate of phosphorus at 1,050 °C was comparatively smaller in the presence of CaCl2. If iron flocculants are used to capture P in wastewater management, it may be necessary to pay special attention to the amount of addition and incineration temperature to maximize the economic potential of recycling.
Subject(s)
Phosphorus , Sewage , Calcium Chloride , Biological Availability , Fertilizers , IncinerationABSTRACT
OBJECTIVE: Ovarian cancer is a deadly gynecologic malignancy with a poor prognosis. It is essential to evaluate the early detection and screening programs of ovarian cancer via timely assessment of long-time survival, particularly in China where those data are incredibly limited. Here, we aimed to provide timely and accurately assessment of long-term survival estimate of ovarian cancer patients from eastern China. METHODS: Data of 770 ovarian cancer patients diagnosed between 2004-2018 were obtained from four cancer registries in Taizhou, eastern China, were included. We used period analysis to calculate five-year relative survival (RS) of aforementioned ovarian cancer patients for overall and the stratification by age at diagnosis and region. RESULTS: Our findings demonstrated that the overall five-year RS for ovarian cancer patients in Taizhou between 2014 and 2018 was 69.2%, while urban areas were higher compared to rural areas (77.6% vs. 64.9%). We also observed a significant age gradient with the five-year RS decreasing from 79.6% for age group < 55 years to 66.9% for age group > 74 years. Furthermore, we identified a clear upward trend of five-year RS over the study period, both overall and stratified by region and age at diagnosis. CONCLUSION: This is the first study in China using period analysis to provide the most up-to-date five-year RS for ovarian cancer patients from Taizhou, eastern China, which reaches 69.2% during 2014-2018. Our results provide valuable information for timely assessment of early detection and screening programs for ovarian cancer in eastern China.
ABSTRACT
The preparation of micro/nano periodic surface structures using femtosecond laser machining technology has been the academic frontier and hotspot in recent years. The formation and evolution of micro/nano periodic ripples were investigated on 2205 stainless steel machined by femtosecond laser. Using single spot irradiation with fixed laser fluences and various pulse numbers, typical ripples, including nano HSFLs (â), nano LSFLs (⟂), nano HSFLs (⟂) and micro grooves (â), were generated one after another in one test. The morphologies of the ripples were analyzed, and the underlying mechanisms were discussed. It was found that the nano holes/pits presented at all stages could have played a key role in the formation and evolution of micro/nano periodic ripples. A new kind of microstructure, named the pea pod-like structure here, was discovered, and it was suggested that the formation and evolution of the micro/nano periodic ripples could be well explained by the pea pod-like structure model.
ABSTRACT
The machining of micro/nano periodic surface structures using a femtosecond laser has been an academic frontier and hotspot in recent years. With an ultrahigh laser fluence and an ultrashort pulse duration, femtosecond laser machining shows unique advantages in material processing. It can process almost any material and can greatly improve the processing accuracy with a minimum machining size and heat-affected zone. Meanwhile, it can fabricate a variety of micro/nano periodic surface structures and then change a material's surface performance dramatically, such as the material's wetting performance, corrosive properties, friction properties, and optical properties, demonstrating great application potential in defense, medical, high-end manufacturing, and many other fields. In recent years, the research is gradually deepening from the basic theory to optimization design, intelligent control, and application technology. Nowadays, while focusing on metal structure materials, especially on stainless steel, research institutions in the field of micro and nano manufacturing have conducted systematic and in-depth experimental research using different experimental environments and laser-processing parameters. They have prepared various surface structures with different morphologies and periods with sound performance, and are one step closer to many civilian engineering applications. This paper reviews the study of micro/nano periodic surface structures and the performance of stainless steel machined using a femtosecond laser, obtains the general evolution law of surface structure and performance with the femtosecond laser parameters, points out several key technical challenges for future study, and provides a useful reference for the engineering research and application of femtosecond laser micro/nano processing technology.
ABSTRACT
Phosphorus is a depletable resource, and the consumption of phosphorus fertilizer increases with the growing population size. Phosphorus recycled from incinerated sludge ash can be a complement to phosphatic fertilizers in districts suffering from phosphorus resource shortages (e.g., Germany, Japan, and Sweden). The apatite inorganic phosphorus (AP) content in incinerated sludge ash is a key factor influencing the recoverability and bioavailability. Biomass straw is rich in calcium and magnesium minerals and can be used as an additive to be blended with sludge to increase the AP content. However, most of the current studies added excessive amounts of calcium-based or biomass additives, and the bioavailability of various Ca-Mg-P minerals generated after the addition of biomass has not been systematically discussed. In this study, the changes of the phosphorus form in the mixed sludge and biomass with Ca/P in the range of 1.0-2.5 are studied, and the influence of temperature and additives on the phosphorus form and the bioavailability of phosphorus in the ash samples are discussed by combining X-ray diffraction and citric acid (CA) leaching experiments. The AP content is very low in the residue of the sludge or corn straw (CS) that has been burned individually. The sludge and the blended sludge and CS were incinerated at various temperatures. As the incineration temperature increased, the conversion of non-apatite inorganic phosphorus (NAIP) to AP was promoted, but the bioavailability did not change until 1050 °C for samples with a Ca/P of 2.5. In the range from 750 to 950 °C, higher temperature promotes the formation of Ca2P2O7 and CaP2O6. CaP2O6 is insoluble in CA; thus, the bioavailability changes little from 750 to 950 °C, although the AP content increases. With the increase of Ca/P, the conversion of NAIP to AP and the bioavailability of phosphorus were promoted. For the blended sludge and CS ash, Ca7Mg2P6O24 appears at 950 and 1050 °C and the bioavailability also increases.
ABSTRACT
To develop high-performance microwave absorption materials with the features of lightweight, thin thickness, broad bandwidth, and strong absorption, an ultralight Ni3S2@N, S-codoped graphene aerogel with a density of 13.5 mg/cm3 has been fabricated by the use of metal-organic frameworks (MOFs) to directly initiate the gelation of graphene oxide strategy. In such a strategy, dual-functional 1D Ni-MOF nanorods not only act as the gelation agent but also afford the doping elements (N and S) originated from the organic species and the precursor for metal sulfide. Due to the synergistic effects of good impedance matching and multiple losses, the optimal reflection loss (RL) of as-prepared Ni3S2@N, S-codoped graphene aerogel reaches -46.9 dB at 17.1 GHz with only 2.0 mm and ultralow filling content (1.75 wt%). The maximum effective absorption bandwidth (EAB) reaches 6.3 GHz (11.7-18.0 GHz) at 2.38 mm, covering the whole Ku band. Moreover, the value of EAB with the RL less than -30 dB can be tuned to 12.2 GHz (5.8-18 GHz) at the absorber thickness ranging from 1.9 to 5.0 mm. This work provides insight for rational design and fabrication of multicomponent-containing graphene aerogels, showing the potential application in lightweight and high-performance microwave absorption.
ABSTRACT
Tight junction (TJ) is a "zippering up" junction structure located at the uppermost portion of adjacent epithelial/endothelial cells in organs and tissues. TJs maintain the relative stability of intracellular substances and functions by closing or opening intercellular pathways, coordinating the entry and exit of molecules of different sizes and charges, and regulating the permeability of paracellular barrier. TJs also prevent microbial invasion, maintain epithelial/endothelial cell polarity, and regulate cell proliferation. TJs are widely present in the skin and mucosal epithelial barriers, intestinal epithelial barrier, glomerular filtration barrier, bladder epithelial barrier, blood-brain barrier, brain-blood tumor barrier, and blood-testis barrier. TJ dysfunction in different organs can lead to a variety of diseases. In addition to signal pathways, transcription factors, DNA methylation, histone modification, TJ proteins can also be regulated by a variety of non-coding RNAs, such as micro-RNAs, long-noncoding RNAs, and circular RNAs, directly or indirectly. This review summarizes the structure of TJs and introduces the functions and regulatory mechanisms of TJs in different organs and tissues. The roles and mechanisms of non-coding RNAs in the regulation of TJs are also highlighted in this review.
Subject(s)
RNA, Untranslated/physiology , Tight Junction Proteins/physiology , Tight Junctions/physiology , Actin Cytoskeleton/physiology , Animals , Blood-Brain Barrier , Blood-Testis Barrier , Humans , Intestinal Mucosa/physiologyABSTRACT
Competing endogenous RNAs (ceRNAs), including long noncoding RNA (lncRNA), circular RNA (circRNA), pseudogenes, synthetic miRNA inhibitors, etc. are classes of RNAs that can compete and interact with each other within an organism. There are regions in these RNAs that can be bound by messenger-RNA-interfering complementary RNA (microRNA), called microRNA response elements (MREs). These RNAs compete with each other to combine complementary microRNAs and MREs to form ceRNA regulatory mechanisms and participate in the regulation of many biological processes. The oxidative stress injury of melanocytes is one of the crucial mechanisms of vitiligo. However, it is unclear whether the ceRNA regulation mechanism is involved in the oxidative stress injury of melanocytes. The purpose of this study is to explore the changes of messenger RNA (mRNA), lncRNAs, and circRNAs in melanocytes under oxidative stress and to identify the key ceRNA regulatory networks. Compared with the normal cells, the chip detection of ceRNA expression profile showed that the expression of 491 mRNAs, 865 lncRNAs, and 1161 circRNAs were altered more than fivefold during the oxidative stress injury of melanocytes. The oxidative stress-related genes (SOD2, PTGS2, DHFR, HMOX1, FOSL1, and PARP1), cell cycle-related genes (CDK1, CCNB1, CCNA2, OIP5, and MK167), and apoptosis-related gene (BIRC5) were identified in the formation of ceRNA regulation networks with lncRNAs and circRNAs, which shares the common MREs. Further verification found that LNCV6_120941_PI430048170 or hsa_circ_0048910 might regulate the expression of SOD2 by sponging hsa-miR-4755-3p, LNCV6_119109_PI430048170, or hsa_circ_0048909 might regulate the expression of HMOX1 by sponging hsa-miR-6721-5p in the oxidative stress injury of melanocytes. In conclusion, complex changes of the ceRNA regulatory network in the oxidative stress response of melanocytes are evident. Oxidative stress may mediate melanocyte injury through the ceRNA regulation mechanism and induce the pathogenesis of vitiligo.
Subject(s)
Gene Regulatory Networks , Melanocytes/metabolism , Oxidative Stress/genetics , RNA/genetics , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle/genetics , Cell Cycle/radiation effects , Gene Ontology , Gene Regulatory Networks/drug effects , Humans , Hydrogen Peroxide/pharmacology , Melanocytes/cytology , Melanocytes/drug effects , Oxidative Stress/drug effectsABSTRACT
The occurrence of hyperpigmentation or hypopigmentation after inflammation is a common condition in dermatology and cosmetology. Since the exact mechanism of its occurrence is not yet known, prevention and treatment are troublesome. Previous studies have confirmed that αmelanocytestimulating hormone, stem cell factor and other factors can promote melanogenesisrelated gene expression through the activation of signaling pathways. Recent studies have revealed that a variety of inflammatory mediators can also participate in the regulation of melanogenesis in melanocytes. In this review, we summarized that interleukin18, interleukin33, granulocytemacrophage colony stimulating factor, interferonγ, prostaglandin E2 have the effect of promoting melanogenesis, while interleukin1, interleukin4, interleukin6, interleukin17 and tumor necrosis factor can inhibit melanogenesis. Further studies have found that these inflammatory factors may activate or inhibit melanogenesisrelated signaling pathways (such as protein kinase A and mitogen activated protein kinase) by binding to corresponding receptors, thereby promoting or inhibiting the expression of melanogenesisrelated genes and regulating skin pigmentation processes. This suggests that the development of drugs or treatment methods from the perspective of regulating inflammation can provide new ideas and new targets for the treatment of pigmented dermatosis. This review outlines the current understanding of the inflammation factors' roles in melanogenesis.
Subject(s)
Inflammation , Melanins/biosynthesis , Pigmentation Disorders/therapy , Signal Transduction , Skin Pigmentation/immunology , Cytokines/immunology , Dinoprostone/metabolism , Humans , Melanocytes/immunology , Melanocytes/metabolism , Pigmentation Disorders/immunology , alpha-MSH/metabolismABSTRACT
As a main part of pigmentation disorders, skin depigmentation diseases such as vitiligo and achromic naevus are very common and get more attention now. The pathogenesis of depigmentation includes melanocyte dysfunction and loss, which are possibly caused by heredity, autoimmunity and oxidative stress. Among them, oxidative stress plays a key role; however, few clinical treatments can deal with oxidative stress. As reported, Cistanche deserticola polysaccharide (CDP) is an effective antioxidant; based on that, we evaluated its role in melanocyte and further revealed the mechanisms. In this study, we found that CDP could promote melanogenesis in human epidermal melanocytes (HEMs) and mouse melanoma B16F10 cells, it also induced pigmentation in zebrafish. Furthermore, CDP could activate mitogen-activated protein kinase (MAPK) signal pathway, then up-regulated the expression of microphthalmia-associated transcription factor (MITF) and downstream genes TYR, TRP1, TRP2 and RAB27A. Otherwise, we found that CDP could attenuate H2 O2 -induced cytotoxicity and apoptosis in melanocytes. Further evidence revealed that CDP could enhance NRF2/HO-1 antioxidant pathway and scavenge intracellular ROS. In summary, CDP can promote melanogenesis and prevent melanocytes from oxidative stress injury, suggesting that CDP helps maintain the normal status of melanocytes. Thus, CDP may be a novel drug for the treatment of depigmentation diseases.
Subject(s)
Cistanche/chemistry , Heme Oxygenase-1/genetics , Melanins/genetics , Membrane Proteins/genetics , NF-E2-Related Factor 2/genetics , Polysaccharides/pharmacology , Animals , Humans , Melanins/antagonists & inhibitors , Melanins/biosynthesis , Melanocytes/drug effects , Melanoma, Experimental/drug therapy , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Mice , Oxidative Stress/drug effects , Pigmentation/drug effects , Pigmentation/genetics , Pigmentation Disorders/drug therapy , Pigmentation Disorders/genetics , Pigmentation Disorders/pathology , Polysaccharides/chemistry , Zebrafish/geneticsABSTRACT
BACKGROUND/AIMS: This study aimed to examine antioxidants in patients with acute kidney injury (AKI) and determine whether serum protein thiol levels are associated with all-cause 90-day mortality in patients with hospital-acquired AKI. METHODS: According to the RIFLE criteria, 160 patients with hospital-acquired AKI were enrolled in our prospective cohort study. As controls, 72 critically ill patients without AKI and 72 age and sex-matched healthy subjects were also recruited. Serum protein thiol levels were analyzed in relation to all-cause mortality of patients with AKI. RESULTS: Serum protein thiol levels in AKI patients were lower than those in healthy people (p=0.010). Protein thiol levels showed a weak but significant positive correlation with serum albumin levels. The 90-day overall mortality rate was higher in AKI patients with high serum protein thiol levels than in those with low levels (p=0.032 by log rank test). In multivariate analysis (Cox regression), serum protein thiol levels (p=0.031) were independently associated with 90-day overall mortality after adjustment for age, sex, sepsis, and the Acute Physiology and Chronic Health Evaluation II score. CONCLUSIONS: Patients with hospital-acquired AKI have remarkably low serum protein thiol levels. Elevated protein thiol levels are associated with 90-day overall mortality in hospital-acquired AKI.
Subject(s)
Acute Kidney Injury/blood , Acute Kidney Injury/mortality , Blood Proteins/analysis , Iatrogenic Disease , Sulfhydryl Compounds/blood , APACHE , Adult , Aged , China/epidemiology , Cohort Studies , Critical Illness/mortality , Female , Hospital Mortality , Humans , Male , Middle Aged , Prospective Studies , Risk Assessment , Serum Albumin/analysis , Survival Analysis , Treatment OutcomeABSTRACT
We compared changes in serum cystatin C (Cys C) and creatinine (sCr) levels for detecting contrast-induced acute kidney injury; 350 consecutive patients who underwent peripheral arterial angiography were prospectively enrolled. Serum Cys C and sCr levels were assayed at predefined time points after contrast-media exposure. During 1-year follow-up, major adverse events (MAEs) including all-cause mortality and dialysis were assessed. A sCr increase ≥25% was not associated with MAEs, whereas a serum Cys C increase ≥5% at 24 hours was associated with higher probability of MAEs (P = .010). The independent predictors of 1-year MAEs were older age (P = .004), lower prealbumin levels (P = .022), and serum Cys C increase ≥5%. In patients who underwent peripheral angiography, a serum Cys C increase ≥5% was an independent predictor of 1-year MAEs.
Subject(s)
Acute Kidney Injury/blood , Acute Kidney Injury/chemically induced , Contrast Media/adverse effects , Creatinine/blood , Cystatin C/metabolism , Peripheral Arterial Disease/diagnostic imaging , Acute Kidney Injury/diagnosis , Acute Kidney Injury/mortality , Acute Kidney Injury/therapy , Age Factors , Aged , Aged, 80 and over , Biomarkers/blood , China , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Peripheral Arterial Disease/blood , Peripheral Arterial Disease/mortality , Prealbumin/metabolism , Predictive Value of Tests , Prognosis , Proportional Hazards Models , Prospective Studies , Radiography , Renal Dialysis , Risk Factors , Time FactorsABSTRACT
The arabinogalactan (AG) of the mycobacterial cell wall consists of an arabinan region, a galactan region and a disaccharide linker. Decaprenylphosphoryl-D-arabinose (DPA) is the donor for arabinofuran residues, which are formed from phosphoribose diphosphate (PRPP) and decaprenyl phosphate (DP). DP is sequentially catalyzed by a three-step process that involves a transferase, a phosphatase and an epimerase. Rv3807c is a putative phospholipid phosphatase that might generate the intermediate product of decaprenyl-phosphoryl-ribose (DPR) in DPA biosynthesis. Mycobacterium smegmatis MSMEG_6402 is a homolog gene of Mycobacterium tuberculosis Rv3807c and was substituted for the functional identification of Rv3807c. Previously, we generated a conditional MSMEG_6402 gene knockout strain (M. sm-ΔM_6402) that exhibited significantly affected cell wall structure. To understand the function of MSMEG_6402 in DPA biosynthesis, this gene was amplified and expressed, and the resulting protein was identified and purified using a His-tagged approach. A MSMEG_6402 enzymatic reaction system with PRPP and DP as substrates was utilized, and the reaction products were separated using thin layer chromatography (TLC). The results revealed a specific lipid-linked sugar band that appeared in the reaction with the addition of MSMEG_6402. Furthermore, ESI-MS detection was utilized in this study, and the results revealed that the enzymatic reaction products involving MSMEG_6402 included DPPR and a sodium ion adduct of DPR. Additionally, the phosphatase activity of MSMEG_6402 was also determined through phosphate group detection using the colorimetric method. Based on our results together with the results of previous studies, including the functional identification and bioinformatics analysis of M. tuberculosis Rv3807c, we propose that MSMEG_6402, as a phosphatase, has an intimate relationship with DPA biosynthesis.
Subject(s)
Arabinose/analogs & derivatives , Bacterial Proteins/metabolism , Biosynthetic Pathways/genetics , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/metabolism , Phosphoric Monoester Hydrolases/metabolism , Arabinose/biosynthesis , Bacterial Proteins/genetics , Chromatography, Thin Layer , Cloning, Molecular , Colorimetry , Gene Deletion , Gene Expression , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Phosphoric Monoester Hydrolases/genetics , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , TerpenesABSTRACT
Decaprenylphosphoryl-d-arabinofuranosyl (DPA), the immediate donor for the polymerized d-Araf residues of mycobacterial arabinan, is synthesized from 5-phosphoribose-1-diphosphate (PRPP) in three-step reactions. (i) PRPP is transferred to decaprenyl-phosphate (DP) to form decaprenylphosphoryl-d-5-phosphoribose (DPPR). (ii) DPPR is dephosphorylated to form decaprenylphosphoryl-d-ribose (DPR). (iii) DPR is formed to DPA by the epimerase. Mycobacterium tuberculosis Rv3806c and heteromeric Rv3790/Rv3791 have been identified as the PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase and the epimerase respectively. Rv3807c, however, as the candidate of phospholipid phosphatase, catalyzing the biosynthesis of decapreny-l-phosphoryl-ribose (DPR) from decaprenylphosphoryl-ß-d-5-phosphoribose by dephosphorylating, has no direct experimental evidence of its essentiality in any species of mycobacterium. In this study, Rv3807c gene was amplified from the genome of M. tuberculosis H37Rv by PCR, and was successfully expressed in Escherichia coli BL21 (DE3) via the recombinant plasmid pColdII-Rv3807c. The resulting protein with the 6× His-tag was identified by SDS-PAGE and Western blotting. The protein was predicted through bioinformatics to contain three transmembrane domains, the N-terminal peptide, and a core structure with phosphatidic acid phosphatase type2/haloperoxidase. This study provides biochemical and bioinformatics evidence for the importance of Rv3807c in mycobacteria, and further functional studies will be conducted for validating Rv3807c as a promising phospholipid phosphatase in the synthetic pathway of DPA.
ABSTRACT
Arabinogalactan (AG) of mycobacterial cell wall consists of arabinan region, galactan region and disaccharide linker. The arabinan is composed of D-arabinofuranose residues, and decaprenyphosphoryl-D-arabinose (DPA) is the donor of the D-arabinofuranose residues. DPA is formed from phosphoribose diphosphate (PRPP) in a four-step process catalyzed by transferase, phosphatase and epimerase, respectively. Mycobacterium tuberculosis Rv3806c has been identified as PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase, and heteromeric Rv3790/Rv3791 has epimerase activity. Rv3807c is putative phospholipid phosphatase. However, there is no direct biochemical evidence since expression of Rv3807c has been unsuccessful. Mycobacterium smegmatis MSMEG_6402 is ortholog of Rv3807c. To investigate the function of MSMEG_6402 on AG biosynthesis, a conditional MSMEG_6402 gene knock out (M. sm-ΔM_6402) strain was constructed through homologous recombination technique. The morphological and compositional changes of cell wall were examined in the M. sm-ΔM_6402 strain. The M. sm-ΔM_6402 strain grew at non-permissive temperature slower than that at permissive temperature, indicating that MSMEG_6402 is non-essential for growth of M. smegmatis. The change of cell shape and detectable bulging on the cell surface of M. sm-ΔM_6402 strain were observed by scanning electron microscopy, and curled as well as deformed cell wall of M. sm-ΔM_6402 strain was revealed by transmission electron microscopy. Analysis of sugar composition in the cell wall by HPLC indicated that the ratio of arabinofuran to galactofuran in M. sm-ΔM_6402 strain was changed to 1.7:1 comparing with 2:1 in the wild type. It demonstrates that the lacking MSMEG_6402 interferes the biosynthesis of arabinan. Analyzing 5' P-DPR and DPR from both M. sm-ΔM_6402 strain and wild type M. smegmatis is undergoing in this lab.