Dapsone is an important medication for the treatment of leprosy, but a life-threatening drug hypersensitivity syndrome develops in some patients. In this report from China, an
HLA-B
locus is ...identified as a strong genetic risk factor for the syndrome.
Dapsone (4-4′-sulfonyldianiline), which was first synthesized in 1908,
1
is both an antibiotic and an antiinflammatory agent. Dapsone alone or in combination with other drugs has been used for the prevention and treatment of infectious diseases (e.g., leprosy, malaria, and actinomycetoma, as well as
Pneumocystis jirovecii
pneumonia in persons with human immunodeficiency virus HIV infection) and chronic inflammatory diseases characterized by the infiltration of neutrophils or eosinophils (e.g., dermatitis herpetiformis, linear IgA dermatosis, subcorneal pustular dermatosis, and erythema elevatum diutinum).
2
,
3
About 0.5 to 3.6% of persons treated with dapsone have a drug hypersensitivity syndrome,
3
–
5
which was first described by . . .
Objectives
The impact of electrolyte imbalance on clinical outcomes after acute ischemic stroke (AIS) is still not understood. We investigated the association between hypochloremia and hyponatremia ...upon hospital admission and in‐hospital mortality in AIS patients.
Materials and methods
A total of 3314 AIS patients enrolled from December 2013 to May 2014 across 22 hospitals in Suzhou city were included in this study. Hypochloremia was defined as having a serum chloride concentration <98 mmol/L and hyponatremia as having a serum sodium concentration <135 mmol/L. The Cox proportional hazard model was used to examine the effect of hypochloremia and hyponatremia on all‐cause in‐hospital mortality in AIS patients.
Results
During hospitalization, 118 patients (3.6%) died from all causes. Multivariable model adjusted for age, sex, baseline National Institutes of Health Stroke Scale score, serum sodium, and other potential covariates showed that hypochloremia was associated with a 2.43‐fold increase in the risk of in‐hospital mortality (hazard ratio HR 2.43; 95% confidence interval CI, 1.41‐4.19; P=.001). However, no significant association between hyponatremia (P=.905) and in‐hospital mortality was observed. Moreover, the multivariable analysis found that serum chloride (HR=0.92, 95% CI 0.88‐0.98; P=.004) but not serum sodium (P=.102) was significantly associated with in‐hospital mortality.
Conclusions
Hypochloremia at admission was independently associated with in‐hospital mortality in AIS patients.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The flavonoid naringenin is abundantly present in pomelo peels, and the unprocessed naringenin in wastes is not friendly for the environment once discarded directly. Fortunately, the hydroxylated ...product of eriodictyol from naringenin exhibits remarkable antioxidant and anticancer properties. The P450s was suggested promising for the bioconversion of the flavonoids, but less naturally existed P450s show hydroxylation activity to C3′ of the naringenin. By well analyzing the catalytic mechanism and the conformations of the naringenin in P450, we proposed that the intermediate Cmpd I ((porphyrin)Fe = O) is more reasonable as key conformation for the hydrolyzation, and the distance between C3′/C5′ of naringenin to the O atom of CmpdI determines the hydroxylating activity for the naringenin. Thus, the “flying kite model” that gradually drags the C-H bond of the substrate to the O atom of CmpdI was put forward for rational design. With ab initio design, we successfully endowed the self-sufficient P450-BM3 hydroxylic activity to naringenin and obtained mutant M5-5, with
k
cat
,
K
m
, and
k
cat
/
K
m
values of 230.45 min
−1
, 310.48 µM, and 0.742 min
−1
µM
−1
, respectively. Furthermore, the mutant M4186 was screened with
k
cat
/
K
m
of 4.28-fold highly improved than the reported M13. The M4186 also exhibited 62.57% yield of eriodictyol, more suitable for the industrial application. This study provided a theoretical guide for the rational design of P450s to the nonnative compounds.
Key points
•
The compound I is proposed as the starting point for the rational design of the P450BM3
•
“Flying kite model” is proposed based on the distance between O of Cmpd I and C3′/C5′ of naringenin
•
Mutant M15-5 with 1.6-fold of activity than M13 was obtained by ab initio modification
Graphical abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to ...be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a “near-attack conformation” of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the k cat value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the k cat/K M increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Cytochrome P450 BM3 (BM3) is an important oxidoreductase that is widely used in drug synthesis, chemical synthesis, and other industries. However, as BM3 unquestionably increases costs by consuming a ...natural cofactor that unstably provides electrons, an alternative biomimetic cofactor with simpler structures represented by nicotinamide mononucleotide (NMNH) has been utilized. Currently, few reports exist on artificially modified BM3 enzymes using NMNH, especially regarding theoretical simulation and calculation. With the cognition of the mechanism in mind, we propose a strategy that optimizes and refines catalytic conformation. Based on constrained molecular dynamics simulation, the distance between N-5 of FAD flavin and C-4 of NMNH is used as a cue for the determination of improved conformation, and the potential positive mutants are subsequently screened virtually in accordance with binding free energy requirements. As a result, the
K
cat
/
K
M
values of the favorable mutant S848R increased to 205.38% compared to the wild-type BM3 with NMNH. These data indicate that our strategy can be applied for the specific utilization of biomimetic cofactors by oxidoreductases represented by BM3.
A rational design strategy was proposed to improve the efficient utilization of alternative biomimetic cofactor by P450 BM3 enzyme.
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IJS, KILJ, NUK, UL, UM, UPUK
The discovery of unique substrates is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming, and ...expensive. Although in silico structure-based approaches show great promise, recent extensive studies have shown that these approaches remain a formidable challenge for current biocomputational methodologies. Here we present an open-source, extensible, and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of an enzyme on the basis of the enzyme’s catalysis mechanism. On the basis of a generalized transition state theory of enzyme catalysis, we introduce a modified docking procedure called “mechanism-based restricted docking” (MBRD) for novel substrate recognition from molecular docking. Comprising a series of utilities written in C/Python, THEMIS automatically executes parallel-computing MBRD tasks and evaluates the results with various molecular mechanics (MM) criteria such as energy, distance, angle, and dihedral angle to help identify desired substrates. Exhaustive sampling and statistical measures were used to improve the robustness and reproducibility of the method. We used Candida antarctica lipase B (CALB) as a test system to demonstrate the effectiveness of our computational prediction of (non)substrates. A novel MM score function for CALB substrate identification derived from the near-attack conformation was used to evaluate the possibility of chemical transformation. A highly positive rate of 93.4% was achieved from a CALB substrate library with 61 known substrates and 35 nonsubstrates, and the screening rate has reached 103 compounds/day (96 CPU cores, 100 samples/compound). The performance shows that the present method is perhaps the first reported scheme to meet the requirement for practical applicability to enzyme studies. An additional study was performed to validate the universality of our method. In this verification we employed two distinct enzymes, nitrilase Nit6803 and SDR Gox2181, where the correct rates of both enzymes exceeded 90%. The source code used will be released under the GNU General Public License (GPLv3) and will be free to download. We believe that the present method will provide new insights into enzyme research and accelerate the development of novel enzyme applications.
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IJS, KILJ, NUK, PNG, UL, UM
The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel ...substrates. This article reports an in silico structure‐based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment‐based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo‐keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Food‐derived angiotensin I‐converting enzyme (ACE) inhibitory peptides represent a potential source of new antihypertensive. However, their characteristics and binding mechanisms were not well ...understood. In this study, novel energy calculation and experimentation were combined to elucidate the characteristics and mechanisms of ACE inhibitory tripeptides. ACE inhibitory activity of all 8,000 tripeptides was investigated by in silico experiments. IC50 values of the five top‐rated tripeptides ranged from 5.86 to 21.84 μM. Five hundred top‐ranked tripeptides were chosen for detailed structure–activity analysis, and a significant preference for aromatic amino acids at both C‐ and N‐terminus was found. By binding free energy analysis of nine representative tripeptides via MM/GBSA, electrostatic energy was found to be the leading energy that contributed to the binding of ACE with its high affinity tripeptides. Besides, S355, V380, and V518, three residues positioned around the classical binding pockets of ACE, also played a key role in ACE's binding. Therefore, for tripeptides, their binding pockets in ACE were redefined. In conclusion, the characteristics of ACE inhibitory peptides were more deeply illustrated by the thorough analysis of all tripeptides. The energy analysis allows a better understanding of the binding mechanisms of ACE inhibitory peptides, which could be used to redesign the ACE inhibitors for stronger inhibitory activity.
The characteristics of C‐ and N‐terminus of ACE inhibitory tripeptides were investigated. Electrostatic energy contributed to the binding of ACE with its high affinity tripeptides.
S355, V380, and V518 played a key role ACE’s binding with tripeptides.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are ...regulated still remain to be elucidated. We describe here a novel murine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro. Remarkably, the expression of a catalytically inactive point mutant of Wwp2 abolishes Oct-4 ubiquitination. Moreover, Wwp2 promotes Oct-4 degradation in the presence of overexpressed ubiquitin. The degradation is blocked by treatment with proteasome inhibitor. Fusion of a single ubiquitin to Oct-4 inactivates its transcriptional activity in a heterologous Oct-4-driven reporter system. Furthermore, overexpression of Wwp2 in embryonic stem cells significantly reduces the Oct-4-transcriptional activities. Collectively, we demonstrate for the first time that Oct-4 can be post-translationally modified by ubiquitination and that this modification dramatically suppresses its transcriptional activity. These results reveal that the functional status of Oct-4, in addition to its expression level, dictates its transcriptional activity, and the results open up a new avenue to understand how Oct-4 defines the fate of embryonic stem cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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