Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

Standardized Tip-Apex Distance (STAD): a modified individualized measurement of cephalic fixator position based on its own femoral head diameter in geriatric intertrochanteric fractures with internal fixation.

OBJECTIVE: To design a standardized Tip-Apex Distance (STAD) and analyze the clinical significance of STAD in predicting cut-out in geriatric intertrochanteric fractures with internal fixation. METHODS: Firstly, we designed STAD according to the rule of TAD. We measured the STAD individually based on its own femoral head diameter (iFHD) instead of the known diameter of the lag screw in calculating TAD, resulting in that the STAD is simply the relative quantitation relationship of iFHD (the times of iFHD). In this study, we assumed that all the iFHD was 6D (1iFHD = 6D, or 1D = 1/6 of iFHD) in order for complete match of the Cleveland zone system, easy comparison of the STAD, and convenient identification for artificial intelligence. Secondly, we calculated and recorded all the STAD of cephalic fixator in 123 eligible ITF patients. Thirdly, we grouped all the ITF patients into the Failure and Non-failure groups according to whether cut-out or not, and analyzed the correlation between the cut-out and the STAD. RESULTS: Cleveland zone, Parker's ratio (AP), TAD, and STAD were associated with the cut-out in univariate analysis. However, only STAD was the independent predictor of the cut-out by multivariate analysis. No cut-out was observed when STAD ≤ 2D (1/3 of iFHD). The Receiver Operating Characteristic (ROC) curve indicated that STAD was a reliable predictor of cut-out, and the best cut-off value of STAD was 2.92D. Cut-out rate increased dramatically when STAD increased, especially when STAD > 3D (1/2 of iFHD). CONCLUSION: Essentially, the STAD is a relative quantitation relationship of iFHD. The STAD is a reliable measurement of cephalic fixator position in predicting cut-out in geriatric ITF patients with single-screw cephalomedullary nail fixations. For avoiding cut-out, the STAD should be no more than a half of iFHD. LEVEL OF EVIDENCE: Level III, Prognostic Study.

Structural and Functional Insights into a Nonheme Iron- and α-Ketoglutarate-Dependent Halogenase That Catalyzes Chlorination of Nucleotide Substrates.

Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical ß-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Risk factors for cut-outs in geriatric intertrochanteric fractures with cephalomedullary nailing after obtaining acceptable reduction: a case-control study.

BACKGROUND: It is irresponsible if we disregard reduction quality to talk about cut-outs in intertrochanteric fractures (ITF) with internal fixation. The aim of this study is to analyze the risk-factors for cut-outs in geriatric ITF with cephalomedullary nailing after obtaining acceptable reduction. METHODS: In order to investigate the risk-factors for cut-outs in geriatric ITF after obtaining acceptable reduction, we retrospectively reviewed 367 patients who underwent cephalomedullary nail for ITF in our department between September 2016 and December 2021. Potential variables including demographic data and radiological parameters (namely the fracture type, Singh index, lateral wall fracture, cephalic nail position, Parker's ratio index, tip-apex-distance (TAD), and calcar-referenced TAD (CalTAD)) were collected. Logistic regression analysis was performed to identify the significant risk factors for cut-outs. RESULTS: One hundred twenty-one patients were suitable for this study. Of the 121 cases, nine cases (7.4%) were observed with cut-out or pending cut-out. We found that Age (adjusted odds ratio (OR) 1.158, 95% confidence interval (CI) 1.016 to 1.318, p = 0.028), lateral wall fracture (adjusted OR 11.07, 95%CI 1.790 to 68.380, p = 0.01), and CalTAD (adjusted OR 1.277, 95%CI 1.005 to 1.622, p = 0.045) were independent risk-factors for cut-outs. CONCLUSIONS: Age, lateral wall fracture and CalTAD are independent risk-factors for cut-outs in geriatric ITF with cephalomedullary nailing after obtaining acceptable reduction. In order to avoid cut-outs, an optimal CalTAD is necessary even obtaining acceptable reduction, especially in the over-aged patients with lateral wall fracture.

Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Structural dissection of unnatural ginsenoside-biosynthetic UDP-glycosyltransferase Bs-YjiC from Bacillus subtilis for substrate promiscuity.

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol-(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.

Joint Salvage and Biological Repair of Massive-Cavity Bone Defects After Extensive Curettage of Campanacci Grade II or III Giant Cell Tumor Around the Knee With Vascularized Fibular Autograft and Cancellous Allograft.

OBJECTIVE: The aim of the study was to report the clinical outcomes of repair of massive-cavity bone defects after extensive curettage of Campanacci grade II or III giant cell tumor (GCT) around knee with vascularized fibular autograft and cancellous allograft. METHODS: There were 12 consecutive patients with Campanacci grade II or III GCT around knee treated in our department between 2004 and 2016. All the patients underwent clinical evaluation, plain radiography, and/or magnetic resonance imaging of the knee right after admission. To preserve their knee function, we repaired the massive-cavity bone defects after extensive curettage of GCT by vascularized fibular autografts and cancellous allograft. All the patients were evaluated through clinical examinations, plain radiography of the knee and chest, and Musculoskeletal Tumor Society (MSTS) scores of the lower extremity in the follow-ups. RESULTS: The follow-up ranged from 1.5 to 12.0 years (mean, 4.2 years). There were no local recurrences or lung metastasis in any of the 12 patients at the last follow-up. Ten patients had no pain or experienced occasional pain, and 9 were able to resume their previous work. The mean range of motion of knee flexion was 117 degrees, and the extension was -6 degrees. The mean MSTS score was 24.7, and a total of 10 patients had excellent or good MSTS scores. CONCLUSIONS: It is reliable to achieve knee joint salvage and repair massive-cavity bone defects after extensive curettage with vascularized fibular autograft and cancellous allograft in patients with Campanacci grade II or III GCT around the knee.

Repair of Small-Size Wound With Achilles Tendon Exposure With Proximal Pedicled Cutaneous Neurovascular Flap.

OBJECTIVE: To investigate the clinical outcomes associated with repairing of small-sized wounds of Achilles tendon exposure with proximal pedicled cutaneous neurovascular flap in the dorsolateral foot. METHODS: After thorough debridement, 16 cases with small-sized wounds of Achilles tendon exposure were repaired by proximal pedicled cutaneous neurovascular flap of the dorsolateral foot, and their clinical outcomes were observed. RESULTS: All the flaps in the 16 cases survived completely, excluding the marginal part necrosis in 1 case, and all the wounds were healed. The 2-point discrimination of the flaps was 14.53 ± 1.55 mm (range, 12-17 mm) in patients without sural nerve injury after 3 to 18 months follow-up. No discomfort was felt in wearing normal shoes by all the 16 patients. CONCLUSIONS: It is reasonable to repair the small-sized wounds of Achilles tendon exposure with proximal pedicled cutaneous neurovascular flap of dorsolateral foot due to its effective repair of the wound, relatively uncomplicated surgery, and had satisfactory healing recovery.

Structure basis of non-structural protein pA151R from African Swine Fever Virus.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded ß-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the ß7-ß8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

Structure of a gut microbial diltiazem-metabolizing enzyme suggests possible substrate binding mode.

When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a ß-barrel subdomain and an α-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The ß-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident.

Structural insights into thebaine synthase 2 catalysis.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.

Crystal structure and proposed mechanism of an enantioselective hydroalkoxylation enzyme from Penicillium herquei.

Hydroalkoxylation is a useful and efficient reaction which generates C-O bond and produces cyclic ethers, the common structural elements of natural products. The dedicative enzyme which can catalyze enantioselective hydroalkoxylation named PhnH was recently identified in the herqueinone biosynthetic gene from Penicillium herquei. It catalyzes addition of a phenol to the terminal olefin on substrate to produce a dihydrobenzofuran. Here, the crystal structure of PhnH is reported and the putative substrate-binding pocket is illustrated. Through docking experiment, possible substrate-binding poses are displayed and the catalytic mechanism is therefore proposed. Our findings form the basis for further studies of enantioselective hydroalkoxylation enzymes.

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.

Structural insight into a novel indole prenyltransferase in hapalindole-type alkaloid biosynthesis.

FamD1 is a novel CloQ/NphB-family indole prenyltransferase which involves in hapalindole-type alkaloid biosynthesis. Here the native FamD1 structure and three protein-ligand complexes are analyzed to investigate the molecular basis of substrate binding and catalysis. FamD1 adopts a typical ABBA architecture of aromatic prenyltransferase, in which the substrate-binding chamber is found in the central ß-barrel. The indole-containing acceptor substrate is bound adjacent to the prenyl donor. Based on the complex structures, a catalytic mechanism of FamD1 is proposed. Functional implications on the sister enzyme FamD2 are also discussed.

"Head-to-Middle" and "Head-to-Tail" cis-Prenyl Transferases: Structure of Isosesquilavandulyl Diphosphate Synthase.

We report the first X-ray crystallographic structure of the "head-to-middle" prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis-prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis-prenyl transferases (e.g. cis-farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain-length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate-Asn-Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin-class antibiotics.

The Crystal Structure of a Class of Cyclases that Catalyze the Cope Rearrangement.

Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily 1 and 2 Stig cyclases, which adopt a ß-sandwich fold like the non-catalytic carbohydrate-binding motif. Several complex structures were also determined of indole-based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H-bond to the indole N and triggers the acid-catalyzed Cope rearrangement. Through analyzing the enzyme-ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement.

Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50.

Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-ß-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-ß-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product.

Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-ß-endoglucanase.

Eukaryotic 1,4-ß-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-ß-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (ß/α)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to -4 - -1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme.

Structure and Function of a "Head-to-Middle" Prenyltransferase: Lavandulyl Diphosphate Synthase.

We report the first X-ray structure of the unique "head-to-middle" monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase.

Moenomycin Biosynthesis: Structure and Mechanism of Action of the Prenyltransferase MoeN5.

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are "bent" and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics.