Zinc-doped and biochar support strategies to enhance the catalytic activity of CuFe2O4 to persulfate for crystal violet degradation.

Sulfate radicals-based Fenton-like technology has placed more emphasis on effectively dealing with the threat of dye wastewater. In this work, the Zn-doped CuFe2O4@biochar composite (Cu0.9Zn0.1Fe2O4@BC) was prepared through the convenient sol-gel pyrolysis process and applied as heterogeneous persulfate (PS) activator for crystal violet (CV) degradation. The crystal morphology and physicochemical properties of Cu0.9Zn0.1Fe2O4@BC were investigated by scanning electron microscope (SEM), X-ray diffractometer (XRD), vibrating sample magnetometer (VSM), Brunauer-Emmett-Teller method (BET), and X-ray photoelectron spectroscopy (XPS). The morphology of the catalyst changed before and after Zn doping. The crystallite size, lattice constant, saturation magnetization, and oxygen vacancy content increased after doping Zn. Compared with CuFe2O4@BC, the CV degradation efficiency of Cu0.9Zn0.1Fe2O4@BC activating PS increased from 87.7 to 96.9%, and the corresponding reaction rate constant increased by about 3.69 times. The effect of experimental conditions was systematically studied on the degradation progress. The degradation efficiency of CV was 91% after five times cycle experiments. Multiple experiments indicated that SO4•-, •OH and O2•- predominated for CV degradation. The degradation mechanism of CV in the Cu0.9Zn0.1Fe2O4@BC/PS system involved both free radical (SO4•-, •OH and O2•-) and non-free radical pathways (electron transfer). The possible degradation pathways were investigated according to the ultra-performance liquid chromatography mass spectrometry (UPLC-MS) analysis of degradation intermediates. The result showed that Cu0.9Zn0.1Fe2O4@BC have an excellent catalyst performance, which provides a new strategy for improving catalytic activity.

Identification of oleoylethanolamide as an endogenous ligand for HIF-3α.

Hypoxia-inducible factors (HIFs) are α/ß heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/ß heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.

Bathroom modifications among community-dwelling older adults who experience falls in the United States: A cross-sectional study.

Falls impose substantial health and economic burdens on older adults. Over half of falls in older adults occur at home, with many involving bathroom areas. Limited information is available on the presence of bathroom modifications for those who experience them. Therefore, we examined factors associated with bathroom modifications among older adults with at least one fall in the United States. We analysed the nationally representative 2016 Medicare Current Beneficiary Survey Public Use File of Medicare beneficiaries aged ≥65 years with ≥1 fall (n = 2,404). A survey-weighted logistic model was used to examine associations between bathroom modifications and factors including socio-demographic characteristics, health-related conditions, and fear of falling. Among Medicare beneficiaries with ≥1 fall, 55.5% had bathroom modifications and 50.1% had repeated falls (≥2 falls). Approximately 40.2% of those with repeated falls had no bathroom modifications. In the adjusted model, non-Hispanic Blacks (odds ratio [OR] = 0.38; p < 0.001) and Hispanics (OR = 0.64; p = 0.039) had lower odds of having bathroom modifications than non-Hispanic Whites. Fear of falling and activities of daily living limitations had incremental impacts on having bathroom modifications. This study highlights the need to improve disparities in bathroom modifications for non-Hispanic Black and Hispanic Medicare beneficiaries, including those with repeated falls. With the aging population and growing number of older minorities in the United States, reducing these disparities is vital for fall prevention efforts and aging-in-place.

Simultaneous Detection of Seven Alternaria Toxins in Mixed Fruit Puree by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled with a Modified QuEChERS.

The presence of Alternaria toxins (ATs) in fruit purees may cause potential harm to the life and health of consumers. As time passes, ATs have become the key detection objects in this kind of food. Based on this, a novel and rapid method was established in this paper for the simultaneous detection of seven ATS (tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene, tentoxin, altenusin, and altertoxin I) in mixed fruit purees using ultra-high performance liquid chromatography-tandem mass spectrometry. The sample was prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method to complete the extraction and clean-up steps in one procedure. In this QuEChERS method, sample was extracted with water and acetonitrile (1.5% formic acid), then salted out with NaCl, separated on an ACQUITY UPLC BEH C18 with gradient elution by using acetonitrile and 0.1% formic acid aqueous as eluent, and detected by UPLC-MS/MS under positive (ESI+) and negative (ESI-) electrospray ionization and MRM models. Results showed that the seven ATs exhibited a good linearity in the concentration range of 0.5-200 ng/mL with R2 > 0.9925, and the limits of detection (LODs) of the instrument were in the range of 0.18-0.53 µg/kg. The average recoveries ranged from 79.5% to 106.7%, with the relative standard deviations (RSDs) no more than 9.78% at spiked levels of 5, 10, and 20 µg/kg for seven ATs. The established method was applied to the determination and analysis of the seven ATs in 80 mixed fruit puree samples. The results showed that ATs were detected in 31 of the 80 samples, and the content of ATs ranged from 1.32 µg/kg to 54.89 µg/kg. Moreover, the content of TeA was the highest in the detected samples (23.32-54.89 µg/kg), while the detection rate of Ten (24/31 samples) was higher than the other ATs. Furthermore, the other five ATs had similar and lower levels of contamination. The method established in this paper is accurate, rapid, simple, sensitive, repeatable, and stable, and can be used for the practical determination of seven ATs in fruit puree or other similar samples. Moreover, this method could provide theory foundation for the establishment of limit standard of ATs and provide a reference for the development of similar detection standard methods in the future.

Characterization of Microbial Community in Cold-Chain Hairtail Fish by High-Throughput Sequencing Technology.

ABSTRACT: High-throughput DNA sequencing with the Illumina MiSeq platform was used to analyze the microbial communities in hairtail (Trichiurus haumela) muscle samples to study the diversity and dynamic changes in these communities during cold-chain circulation of these fish. The richness and diversity of the microbial community in hairtail muscle had a transient decline from 0 to 24 h and decreased after the first rise from 24 to 216 h. The diversity and richness of bacteria in cold-chain hairtail reached maximum at 168 h. The Shannon and Simpson diversity indices of the bacteria were 2.96 and 0.16, respectively, and their ACE and Chao1 richness indices were 254.84 and 155.10, respectively. The dominant bacteria belonged to phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, family Pseudomonadaceae, and genus Pseudomonas, and their relative abundances were 80.52, 72.11, 76.68, 23.25, and 53.50%, respectively. These results provide a basis for exploring how to maintain the freshness and predict the shelf life of hairtail.

Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Tanshinone IIA Can Inhibit Angiotensin II-Induced Proliferation and Autophagy of Vascular Smooth Muscle Cells via Regulating the MAPK Signaling Pathway.

To examine the effect of tanshinone IIA on Angiotensin II (Ang II)-induced proliferation and autophagy in vascular smooth muscle cells (VSMCs) and the related mechanism. VSMCs were treated with Ang II with or without tanshinone IIA (1, 5 and 10 µg/mL), and the proliferation, apoptosis in cells with different treatment were examined by methylthiazolyl tetrazolium (MTT) and flow cytometry methods. Moreover, the expression of autophagy related proteins and mitogen-activated protein kinase (MAPK) signaling molecules were examined by RT-quantitative (q)PCR and Western blot methods. Ang II induced significantly increase in the proliferation and autophagy of VSMCs, and the MAPK signaling was activated. Tanshinone IIA can attenuate Ang II-induced effects via down-regulating the MAPK signaling pathway. Tanshinone IIA can inhibit Ang II-induced proliferation and autophagy of VSMCs via regulating the MAPK signaling pathway.

Bidirectional modulation of HIF-2 activity through chemical ligands.

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.

Structural integration in hypoxia-inducible factors.

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1ß) subunits. Here we describe crystal structures for each of mouse HIF-2α-ARNT and HIF-1α-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α-ARNT and HIF-1α-ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

Long-term outcome after cardioverter-defibrillator implantation in patients with Brugada syndrome / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the long-term outcome of implantable cardioverter-defibrillator (ICD) implantation in Brugada syndrome patients and to explore how to reduce the frequency of ICD nappropriate schocks.</p><p><b>METHODS</b>This study included 14 symptomatic patients (mean age (44.3 ± 8.3) years old; all males) with Brugada syndrome implanted with ICD in our hospital between 1998 and 2012, and these patients were followed up routinely every 6 months. The initial ICD parameters were set according o conventional experience. The ventricular tachycardia (VT) zone was programmed to ventricular rate 150-188 bpm/cycle length (CL) 400-320 ms and the ventricular fibrillation (VF) zone was programmed to ventricular rate ≥ 188 bpm/CL ≤ 320 ms. The total events were recorded by ICD. The ICD parameters revision was made by electrophysiological (EP) experts in case of inappropriate shocks.</p><p><b>RESULTS</b>Patients were followed up for mean (43.0 ± 28.3) months. A total of 297 VF/VT events were recorded by ICD. Electrophysiological experts found that 90% (178/198) episodes were true VF ( CL 130-250 ms) among of 198 VF episodes and 147 VF episodes were terminated by one shock and 21 VF events were terminated by two or more shocks, and the rest 10 VF terminated spontaneously. Only 9% (9/99) VT events were true VT (CL 320-360 ms) among of 99 VT episodes. Eight VT episodes were converted by antitachycardia pacing therapy (ATP) and the other one terminated spontaneously. The rest 90 VT episodes (91%) were supraventricular arrhythmias (SVT, CL 340-390 ms). About 90% inappropriate shocks can be reduced by Wavelet discrimination function and optimal programming (VF zone ventricular rate ≥ 222 bpm/CL ≤ 270 ms and/or VT zone ventricular rate 167-222 bpm/CL 270-360 ms ) according to the characteristics of arrhythmia of individual patient.</p><p><b>CONCLUSION</b>ICD can effectively prevent sudden cardiac death and syncope in high-risk patients with Brugada syndrome. The most common complication is inappropriate shock due to SVT. Optimal ICD programming with Wavelet discrimination function can effectively reduce the frequency of inappropriate shock rate.</p>

Sick sinus syndrome associated with hypopituitarism: a case report and literature review.

Though an association between autoimmune diseases and sick sinus syndrome has been reported, there has been no report on the association of hypopituitarism and sick sinus syndrome. Herein, we provide the first case report of hypopituitarism accompanying sick sinus syndrome in a 51-year-old woman presented to our hospital with syncope due to cardiac arrest. The patient was successfully managed by pacemaker installation and hormone replacement therapy.

Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet ß-cells.

Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in ß-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly.

[Long-term outcomes and factors for predicting ventricular arrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To evaluate factors for predicting ventricular arrhythmia, the clinical effect of drugs on patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), and their long-term outcomes. METHODS: Six patients diagnosed with CPVT underwent a series of electrocardiograms and 24-hour Holter monitoring. ß-blockers were recommended for all patients, while some patients were also prescribed propafenone and 1 patient underwent catheter-based renal sympathetic denervation (RDN). The characteristics of electrocardiogram, arrhythmia and long-term outcomes were monitored. RESULTS: Syncope episodes did not occur any longer in 1 patient on ß-blocker, but recurred in 3 other patients and 2 patients died (one due to his cessation of metoprolol for 3 months). Inverted and/or bifid T waves and abnormal U wave were observed in the precordial leads. T wave alternans was observed in 4 patients in the precordial leads. These abnormal electrocardiogram features disappeared or diminished with ß-blocker treatment. All spontaneous episodes of ventricular tachycardia occurred prior to sinus tachycardia and frequent polymorphic premature ventricular contractions. CONCLUSIONS: Bifid and/or inverted T waves, T wave alternans and abnormal U waves together with sinus tachycardia and frequent premature ventricular contractions are indicator for predicting ventricular arrhythmia and assessing the effect of ß-blockers. Compliance with ß-blocker treatment is a strong indicator of outcome.

Serum miR-210 and miR-30a expressions tend to revert to fetal levels in Chinese adult patients with chronic heart failure.

BACKGROUND: MicroRNAs (miRNAs) are widely involved in the process of chronic heart failure (HF), which is characterized by reactivation of the fetal gene program. Here, we examined whether the serum expression levels of some HF-related miRNAs in adult HF patients would tend to revert to fetal levels. METHODS AND RESULTS: Serum was obtained from the peripheral venous blood of 22 HF patients, 18 asymptomatic controls, and the umbilical venous blood of 9 fetuses from 9 independent parturitions. Serum pools of the three groups were initially screened against 40 known HF-associated miRNAs via quantitative reverse transcriptase polymerase chain reaction. Twenty-seven miRNAs were stably expressed in the serum pools. Nine miRNAs showed similar expression levels in the HF and fetus groups compared to the controls, two of which (miR-210, miR-30a) were significantly up-regulated in both groups. These miRNAs showed high diagnostic accuracy and correlations with blood N-terminal prohormone of brain natriuretic peptide, identifying them as potential biomarkers for HF. Putative targets of the miRNAs were predicted with online software programs, and the Kyoto Encyclopedia of Genes and Genomes pathway analysis was employed to identify miRNA-regulated functional modules. In particular, miR-210 seemed to be more closely related than miR-30a to the pathological mechanisms of HF, including the calcium signaling, vascular smooth muscle contraction, transforming growth factor-ß signaling, and aldosterone-regulated sodium reabsorption pathways. CONCLUSION: The serum expression levels of some HF-related miRNAs in HF patients tended towards fetal levels. Among them, miR-210 and miR-30a were elevated in the HF and fetus groups.

Evidence of a role of inositol polyphosphate 5-phosphatase INPP5E in cilia formation in zebrafish.

Inositol phosphatases are important regulators of cell signaling and membrane trafficking. Mutations in inositol polyphosphate 5-phosphatase, INPP5E, have been identified in Joubert syndrome, a rare congenital disorder characterized by midbrain malformation, retinitis pigmentosa, renal cysts, and polydactyly. Previous studies have implicated primary cilia abnormalities in Joubert syndrome, yet the role of INPP5E in cilia formation is not well understood. In this study, we examined the function of INPP5E in cilia development in zebrafish. Using specific antisense morpholino oligonucleotides to knockdown Inpp5e expression, we observed phenotypes of microphthalmia, pronephros cysts, pericardial effusion, and left-right body axis asymmetry. The Inpp5e morphant zebrafish exhibited shortened and decreased cilia formation in the Kupffer's vesicle and pronephric ducts as compared to controls. Epinephrine-stimulated melanosome trafficking was delayed in the Inpp5e zebrafish morphants. Expression of human INPP5E expression rescued the phenotypic defects in the Inpp5e morphants. Taken together, we showed that INPP5E is critical for the cilia development in zebrafish.

Structural analysis of bengamide derivatives as inhibitors of methionine aminopeptidases.

Natural-product-derived bengamides possess potent antiproliferative activity and target human methionine aminopeptidases (MetAPs) for their cellular effects. Several derivatives were designed, synthesized, and evaluated as MetAP inhibitors. Here, we present four new X-ray structures of human MetAP1 in complex with the inhibitors. Together with the previous structures of bengamide derivatives with human MetAP2 and tubercular MtMetAP1c, analysis of the interactions of these inhibitors at the active site provides structural basis for further modification of these bengamide inhibitors for improved potency and selectivity as anticancer and antibacterial therapeutics.

[Clinical features of six patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To describe the clinical features of 6 patients with catecholaminergic polymorphic ventricular tachycardia. METHODS: Clinical data including signs and symptoms, electrocardiograms, Holter monitoring electrocardiograms and echocardiography was analyzed. Definite diagnosis was made based on the mutations of RYR2 and CASQ2. RESULTS: From July 2002 to March 2010, 6 consecutive patients referred to our center because of syncope [4 males, mean age (13.0 ± 4.2) years] were diagnosed with CPVT by clinical evaluation and genetic testing. Their electrocardiograms showed T waves with notch or bimodal and tall U waves in right chest leads. There was no J wave, no ST-segment deviation, no prolongation or shortening of QT interval. We captured the so-called "bidirectional and(or) polymorphic ventricular tachycardia (bVT and pVT)" in 2 out of 6 patients by ECG, in 5 out of 6 patients by 24-hours Holter monitor, in 3 out of 6 patients by exercise test. All patients received ß blockers and no syncope occurred during the 3 months follow-up after discharge from hospital. CONCLUSIONS: CPVT is an inherited cardiac channelopathy characterized by syncope and(or) sudden death relatived to motion. The ECG shows T wave alteration and tall U wave in right chest leads. The mode of its onset is bVT and(or) pVT, and can be captured by Holter easily. ß blocker is a safe and effective remedy for suppressing its attack.

Structural analysis of inhibition of Mycobacterium tuberculosis methionine aminopeptidase by bengamide derivatives.

Natural product-derived bengamides possess potent antiproliferative activity and target human methionine aminopeptidases for their cellular effects. Using bengamides as a template, several derivatives were designed and synthesized as inhibitors of methionine aminopeptidases of Mycobacterium tuberculosis, and initial antitubercular activity were observed. Here, we present three new X-ray structures of the tubercular enzyme MtMetAP1c in complex with the inhibitors in the Mn(II) form and in the Ni(II) form. All amide moieties of the bengamide derivatives bind to the unique shallow cavity and interact with a flat surface created by His-212 of MtMetAP1c in the Mn(II) form. However, the active site metal has significant influence on the binding mode, because the amide takes a different conformation in the Ni(II) form. The interactions of these inhibitors at the active site provide the structural basis for further modification of these bengamide inhibitors for improved potency and selectivity.

Inhibition of Mycobacterium tuberculosis methionine aminopeptidases by bengamide derivatives.

Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.