Role of PemI in the Staphylococcus aureus PemIK toxin-antitoxin complex: PemI controls PemK by acting as a PemK loop mimic.

Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin-antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between ß1 and ß2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.

Description of Salinimicrobium tongyeongense sp. nov., isolated from seawater.

A Gram-stain-negative, non-motile by gliding and moderately halophilic rod-shaped bacterium HN-2-9-2T was isolated from seawater in Tongyeong, Republic of Korea. The strain grew at concentrations of 0.5‒7 % (w/v) NaCl, at pH 5.5‒8.5 and in a temperature range of 18‒45 °C. HN-2-9-2T shared the highest 16S rRNA gene sequence percentage with Salinimicrobium xinjiangense BH206T (98.2 %). The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) values between HN-2-9-2T and the S. xinjiangense BH206T were 76.0 %, 81.9 % and 19.7 %, respectively. The genome comprised 3 509 958 bp with a DNA G+C content of 43.0%. HN-2-9-2T contained MK-6 as the sole menaquinone. The predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH, iso-C16 : 0, iso-C15 : 1G and summed feature 9, comprising iso-C17 : 1ω6c/C16 : 1 10-methyl. The polar lipids contained phosphatidylethanolamine, one unidentified phospholipid, two unidentified aminolipids, an unidentified glycolipid and six unidentified lipids. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Salinimicrobium, for which the name Salinimicrobium tongyeongense sp. nov. is proposed. The type strain is HN-2-9-2T (=KCTC 82934T=NBRC 115920T).

Description of Brachybacterium sillae sp. nov., a thermophilic bacterium isolated from a hot spring.

The taxonomic position of strain EF45031T, isolated from the Neungam Carbonate hot spring, was examined using the polyphasic taxonomic approach. Strain EF45031T shared the highest percentage of 16S rRNA gene sequence with Brachybacterium nesterenkovii CIP 104813 T (97.7%). The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain EF45031T and the type strains B. nesterenkovii CIP 104813 T and B. phenoliresistens Phenol-AT were 77.0%, 69.15%, 21.9% and 75.73%, 68.81%, 20.5%, respectively. Phylogenomic analysis using an up-to-date bacterial core gene (UBCG) set revealed that strain EF45031T belonged to the genus Brachybacterium. Growth occurred between 25 and 50 ℃ at pH 6.0-9.0 and could tolerate salinity up to 5% (w/v). Strain had anteiso-C15:0 and anteiso-C17:0 as major fatty acids. Menaquinone-7 (MK-7) was the predominant respiratory menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, three aminolipids, and two unidentified glycolipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The genome comprised 2,663,796 bp, with a G + C content of 70.9%. Stress-responsive periplasmic chaperone/protease coding genes were identified in the genome of EF45031T and were not detected in other Brachybacterium species. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Brachybacterium, for which the name Brachybacterium sillae sp. nov. is proposed. The type strain is EF45031T (= KCTC 49702 T = NBRC 115869 T).

Description of Microbacterium neungamense sp. nov. isolated from a hot spring.

The Gram-positive, nonmotile, rod-shaped bacterium EF45044T was isolated from a hot spring in Chungju, South Korea. The strain was able to grow at concentrations of 0‒5% (w/v) NaCl, at pH 6.0‒10.0 and in the temperature range of 18‒50 °C. Strain EF45044T showed the highest 16S rRNA gene sequence similarity (98.2%) with Microbacterium ketosireducens DSM 12510T, and the digital DNA‒DNA hybridization (dDDH), average amino acid identity (AAI), and average nucleotide identity (ANI) values were all lower than the accepted species threshold. Strain EF45044T contained MK‒12 and MK‒13 as the predominant respiratory quinones and anteiso‒C17:0, anteiso‒C15:0, and iso‒C16:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, and glycolipid were detected as the major polar lipids. The cell-wall peptidoglycan contained ornithine. The DNA G + C content was 71.4 mol%. Based on the polyphasic data, strain EF45044T (= KCTC 49703T) presents a novel species of the genus Microbacterium, for which the name Microbacterium neungamense sp. nov. is proposed.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Can preoperative lacrimal endoscopic evaluation change the paradigm of conventional lacrimal surgery?

PURPOSE: To evaluate the effectiveness of preoperative lacrimal endoscopic evaluation (LEE) of lacrimal duct system (LDS). DESIGN: Retrospective comparative case series METHODS: From March 2016 to February 2020, the charts of patients chosen to undergo endoscopic dacryocystorhinostomy (EDCR) or silicone tube intubation (STI) were reviewed retrospectively. Group 1 included patients that underwent EDCR, and group 2 included patients that underwent STI. Preoperative LEE was performed for all patients. In group 1, we compared the functional success rate for patients who had been converted to STI with the patients who had undergone EDCR. In group 2, we compared the functional success rate of STI with those who had had STI without LEE. RESULTS: In group 1, 19 (54.3%) eyes were converted to STI following LEE, and the functional success rate was 84.2%, which is not significantly different from that of the EDCR group following LEE (p = 0.608). The functional success rate of EDCR without LEE was not different from that of STI following LEE (p = 1.000). In group 2, five eyes (26.3%) were converted to EDCR following LEE. The group undergoing STI following LEE showed a significantly higher functional success rate (95.7%) than the group without LEE (66.6%, p = 0.023). CONCLUSION: Preoperative LEE enables direct visualization of the LDS and helps to obtain more accurate diagnosis. This allows for the best surgical option based on LEE findings, which can contribute to better results. Therefore, LEE would be expected to change the paradigm of the classical management of LDS.

Crystal structure of proteolyzed VapBC and DNA-bound VapBC from Salmonella enterica Typhimurium LT2 and VapC as a putative Ca2+ -dependent ribonuclease.

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.

A large-scale metagenomic study for enzyme profiles using the focused identification of the NGS-based definitive enzyme research (FINDER) strategy.

Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next-generation sequencing. Culture-independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of 'focused identification of next-generation sequencing-based definitive enzyme research' enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands-on obtention of high-throughput biological diversity and potential function profiles, without being time-consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.

Morphological changes after lower eyelid epiblepharon surgery in Asian children.

BACKGROUND: This study aimed to determine the morphological changes in Asian lower eyelid epiblepharon patients after surgery. METHODS: The medical records of 59 patients who underwent lower eyelid epiblepharon repair were reviewed retrospectively. Eighty-nine patients who underwent strabismus surgery were set as the control group. The photographs for each group were analyzed based on the following factors: inferior half area (IHA) of the eye, eyelash angular direction (EAD), angle between the eyelashes and the cornea, marginal reflex distance 1 (MRD1) and marginal reflex distance 2 (MRD2). RESULTS: After surgery, the medial EAD changed from 92.45° ± 20.21° (mean ± SD) to 79.43° ± 23.31°, while the central and lateral EADs were unchanged. IHA increased from 36.33 ± 9.78 mm3 to 43.06 ± 10.57 mm3, and MRD1 increased from 1.92 ± 0.99 mm to 2.50 ± 0.93 mm, whereas MRD2 did not change. The mean angle between the eyelashes and the cornea increased from 39.64° to 72.19° immediately postoperatively, but had reduced to 58.75° 3 months later, followed by no further significant change at the 6-month and 9-month postoperative follow-ups. CONCLUSIONS: There is morphological changes of the eyelid after lower eyelid epiblepharon surgery, with increases in the IHA and MRD1. In addition, contact between the eyelashes and the cornea occurred mainly in the medial portion of the eyelid the position, which everted and stabilized over 3 months. Thus, follow-up observations are required for at least 3 months to properly evaluate the surgical outcome.

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

Active Compound of Pharbitis Semen (Pharbitis nil Seeds) Suppressed KRAS-Driven Colorectal Cancer and Restored Muscle Cell Function during Cancer Progression.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven colorectal cancer (CRC) is notorious to target with drugs and has shown ineffective treatment response. The seeds of Pharbitis nil, also known as morning glory, have been used as traditional medicine in East Asia. We focused on whether Pharbitis nil seeds have a suppressive effect on mutated KRAS-driven CRC as well as reserving muscle cell functions during CRC progression. Seeds of Pharbitis nil (Pharbitis semen) were separated by chromatography and the active compound of Pharbitis semen (PN) was purified by HPLC. The compound PN efficiently suppressed the proliferation of mutated KRAS-driven CRC cells and their clonogenic potentials in a concentration-dependent manner. It also induced apoptosis of SW480 human colon cancer cells and cell cycle arrest at the G2/M phase. The CRC related pathways, including RAS/ERK and AKT/mTOR, were assessed and PN reduced the phosphorylation of AKT and mTOR. Furthermore, PN preserved muscle cell proliferation and myotube formation in cancer conditioned media. In summary, PN significantly suppressed mutated KRAS-driven cell growth and reserved muscle cell function. Based on the current study, PN could be considered as a promising starting point for the development of a nature-derived drug against KRAS-mutated CRC progression.

Functional details of the Mycobacterium tuberculosis VapBC26 toxin-antitoxin system based on a structural study: insights into unique binding and antibiotic peptides.

Toxin-antitoxin (TA) systems are essential for bacterial persistence under stressful conditions. In particular, Mycobacterium tuberculosis express VapBC TA genes that encode the stable VapC toxin and the labile VapB antitoxin. Under normal conditions, these proteins interact to form a non-toxic TA complex, but the toxin is activated by release from the antitoxin in response to unfavorable conditions. Here, we present the crystal structure of the M. tuberculosis VapBC26 complex and show that the VapC26 toxin contains a pilus retraction protein (PilT) N-terminal (PIN) domain that is essential for ribonuclease activity and that, the VapB26 antitoxin folds into a ribbon-helix-helix DNA-binding motif at the N-terminus. The active site of VapC26 is sterically blocked by the flexible C-terminal region of VapB26. The C-terminal region of free VapB26 adopts an unfolded conformation but forms a helix upon binding to VapC26. The results of RNase activity assays show that Mg2+ and Mn2+ are essential for the ribonuclease activity of VapC26. As shown in the nuclear magnetic resonance spectra, several residues of VapB26 participate in the specific binding to the promoter region of the VapBC26 operon. In addition, toxin-mimicking peptides were designed that inhibit TA complex formation and thereby increase toxin activity, providing a novel approach to the development of new antibiotics.

Two distinct mechanisms of transcriptional regulation by the redox sensor YodB.

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor.

The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the ribonucleolytic enzyme MazF and has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (EDF). Although it is well established that the increase in EDF-mediated toxicity of MazF drives a cell-killing phenomenon, the molecular details are poorly understood. Moreover, the divergence in sequences among reported EDFs suggests that each bacterial species has a unique EDF. To address these open questions, we report here the structures of MazF4 and MazEF4 complexes from M. tuberculosis, representing the first MazEF structures from this organism. We found that MazF4 possesses a negatively charged MazE4-binding pocket in contrast to the positively charged MazE-binding pockets in homologous MazEF complex structures from other bacteria. Moreover, using NMR spectroscopy and biochemical assays, we unraveled the molecular interactions of MazF4 with its RNA substrate and with a new EDF homolog originating from M. tuberculosis The EDF homolog discovered here possesses a positively charged residue at the C terminus, making this EDF distinct from previously reported EDFs. Overall, our results suggest that M. tuberculosis evolved a unique MazF and EDF and that the distinctive EDF sequence could serve as a starting point for designing new anti-tuberculosis drugs. We therefore conclude that this study might contribute to the development of a new line of anti-tuberculosis agents.

Risk of recurrence after endoscopic resection of early colorectal cancer with positive margins.

BACKGROUND AND STUDY AIM: Additional surgery is recommended if an endoscopically resected T1 colorectal cancer (CRC) specimen shows a positive resection margin. We aimed to investigate the significance of a positive resection margin in endoscopically resected T1 CRC. PATIENTS AND METHODS: We enrolled 265 patients with T1 CRC who underwent endoscopic resection between January 2001 and December 2016. The inclusion criteria were: 1) complete resection by endoscopy, and 2) pathology of a positive margin. Among the 265 patients, 213 underwent additional surgery and 52 did not. In the additional surgery group, various clinicopathological factors were evaluated with respect to the presence or absence of residual tumor. The follow-up results were assessed in the group that did not undergo additional surgery. RESULTS: In the 213 patients who underwent additional surgery, residual tumor was detected in 13 patients (6.1 %), and none of the clinicopathological factors was significantly associated with the presence of residual tumor. Among the 52 patients who did not undergo additional surgery, recurrence was detected in 4 (7.7 %), and all 4 underwent salvage surgery. Among these four patients, three had no risk factors for lymph node metastasis and recurrence was at the previous resection site; pathology was high grade dysplasia, rpT3N0M0, and rpT1N0M0, respectively. CONCLUSIONS: A positive resection margin in endoscopically resected T1 CRC is related to a relatively low incidence of residual tumor (6.1 %). Although current guidelines recommend additional surgery for such cases, surveillance and timely salvage surgery could be another option in selected cases.

Halorubrum aethiopicum sp. nov., an extremely halophilic archaeon isolated from commercial rock salt.

A novel extremely halophilic archaeon, designated SAH-A6T, was isolated from a sample of commercial rock salt in Ethiopia. Cells of SAH-A6T were aerobic and pleomorphic. The strain was able to grow at concentrations of 15-30 % (w/v) NaCl (optimum 20-25 % NaCl), at pH 6.0-9.0 (optimum pH 7.0) and in a temperature range of 30-55 °C (optimum 37-45 °C). Mg2+ was not required for growth of SAH-A6T cells. On the basis of 16S rRNA gene sequence analysis, strain SAH-A6T was closely related to Halorubrum halodurans Cb34T (99.1 %), Halorubrum rubrum YC87T (98.9 %), Halorubrum aquaticum EN-2T (98.7 %), Halorubrum cibi JCM 15757T (98.4 %), Halorubrum luteum CGSA15T (97.3 %), Halorubrum lipolyticum 9-3T (97.1 %), Halorubrum tibetense 8W8T (97.1 %), Halorubrum kocurii JCM 1478T (97.1 %), Halorubrum halophilum B8T (97.0 %) and Halorubrum persicum C49T (97.0 %). Phylogenetic analysis based on the rpoB' gene sequences showed that strain SAH-A6T was closely related to Hrr. halodurans Cb34T (99.7 %), Hrr. aquaticum JCM 14031T (99.3 %) and other members of the genus Halorubrum (<99.0 %). The DNA G+C content of the strain was 68.0 mol%. DNA-DNA hybridization between strain SAH-A6T and the most closely related members of the genus Halorubrum were below 55 %, suggesting that the new isolate constitutes a different genospecies. On the bases of chemotaxonomic, phenotypic and genotypic data, strain SAH-A6T (=KCCM 43215T=JCM 31519T) represents a novel species of the genus Halorubrum, for which the name Halorubrumaethiopicum sp. nov. is proposed.

Feasibility of Selective Lateral Node Dissection Based on Magnetic Resonance Imaging in Rectal Cancer After Preoperative Chemoradiotherapy.

BACKGROUND: Lateral pelvic node metastasis is one of the major causes of local recurrence in advanced rectal cancer after preoperative chemoradiotherapy (CRT). However, lateral pelvic node dissection (LPND) is still a challenging surgical procedure in terms of surgical morbidity. This study aimed to investigate the feasibility and safety of LPND in patients with advanced rectal cancer after preoperative CRT. MATERIALS AND METHODS: Records of 80 consecutive patients who underwent total mesorectal excision (TME) with LPND for initially enlarged lateral pelvic nodes (LPNs) (short-axis diameter ≥5 mm on magnetic resonance imaging before preoperative CRT) between 2011 and 2016 were retrospectively reviewed. Surgical outcomes of these patients were compared with those of 281 patients who underwent TME alone. RESULTS: Ninety-nine LPND procedures were performed, including 19 bilateral LPNDs. Pathologically proven LPN metastasis was identified in 32 (32.3%) LPND cases after preoperative CRT. Multiple (odds ratio = 12.908, 95% confidence interval: 3.355-49.660, P < 0.001) and persistently enlarged LPNs (odds ratio = 27.093, 95% confidence interval: 6.072-120.896, P < 0.001) were independently associated with LPN metastasis. The rates of overall postoperative 30-d morbidity (42.5% versus 32.4%, P = 0.093) and urinary retention (11.3% versus 7.1%, P = 0.230) were similar between the TME with LPND and TME-only groups. CONCLUSIONS: The postoperative morbidity of TME with LPND was comparable with TME-only group. The rate of LPN metastasis remained high after preoperative CRT, especially in patients with multiple or persistently enlarged LPNs.

Comparative Histomorphometric Analysis of Maxillary Sinus Augmentation With Deproteinized Bovine Bone and Demineralized Particulate Human Tooth Graft: An Experimental Study in Rabbits.

PURPOSE: The aim of this animal study is to evaluate, by histomorphometric analysis, new bone formation in rabbit maxillary sinuses with Bio-Oss and demineralized particulate human tooth graft. MATERIALS AND METHODS: Bilateral sinus augmentation procedures were performed in 8 adult male rabbits. After preparation of replaceable bony windows on the lateral wall of the nasal cavity with a piezoelectric surgical device, deproteinized bovine graft (Bio-Oss) was grafted in the new compartment of the maxillary sinus after elevation of the sinus membrane in the control group. In the experimental group, the demineralized human particulate tooth bone was grafted in the sinus. The replaceable bony window was repositioned over the bone graft in both groups. Animals were killed at 2 and 8 weeks after the surgical procedure. The augmented sinuses were evaluated by histomorphometric analysis using hematoxylin-eosin and Masson trichrome stains. RESULTS: Histologically, new bone was revealed along the elevated sinus membrane and both bone grafts. In the control group, the new bone area at 8 weeks was not significantly different than that at 2 weeks. In the experimental group, the new bone area at 8 weeks was significantly greater than that at 2 weeks. CONCLUSION: Significant higher new bone formation was revealed in the experimental group than in the control group.

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity.IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the characterization and functional annotation of a putative nonphosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent, bifunctional, sugar-isomerizing activity. This hyperthermophilic R3E exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from those of its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70°C. Therefore, TM0416 can be functionally classified as a novel type of promiscuous R3E with a potential for the production of rare sugars for the food and pharmaceutical industries.

Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides.

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation.