Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae)

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C₁₈ column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 μM, 50 μM, and 75 μM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 μM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.     

Characterization of antimicrobial peptides from the skin secretions of the Malaysian frogs, Odorrana hosii and Hylarana picturata (Anura:Ranidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from Hose's rock frog Odorrana hosii (Boulenger, 1891) led to the isolation of 8 peptides with differential antibacterial activities. Structural characterization demonstrated that the peptides belonged to the esculentin-1 (1 peptide), esculentin-2 (1 peptide), brevinin-1 (2 peptides), brevinin-2 (2 peptides), and nigrocin-2 (2 peptides) families of antimicrobial peptides. Similar analysis of skin secretions from the Malaysian fire frog Hylarana picturata (Boulenger, 1920) led to the isolation and characterization of peptides belonging to the brevinin-1 (2 peptides), brevinin-2 (5 peptides), and temporin (1 peptide) families. The differences in antimicrobial activities of paralogous peptides provide insight into structure-activity relationships, emphasizing the importance of cationicity in determining potency. The substitution Lys(11)-->Gln in brevinin-1HSa (FLPAVLRVAAKIVPTVFCAISKKC) from O. hosii abolishes growth inhibitory activity against Escherichia coli but has no effect on the high potency (MIC=8mug/ml) against Staphylococcus aureus. In contrast, the substitution (Gly(4)-->Asp) in brevinin-2PTb (GFKGAFKNVMFGIAKSAGKSALNALACKIDKSC) from H. picturata reduces activity against both E. coli and S. aureus. Cladistic analysis based upon the amino acid sequences of the brevinin-2 peptides from Asian frogs provides evidence for sister taxon relationships between O. hosii and O. livida and between H. picturata and H. güntheri.     

Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog,Hydrophylax bahuvistara (Anura: Ranidae)

Two novel peptides (brevinin1 HYba1 and brevinin1 HYba2) were identified from the skin secretion of the frog Hydrophylax bahuvistara, endemic to Western Ghats, India, and their amino acid sequences were confirmed using cDNA cloning and LC/MS/MS. Antibacterial, hemolytic, and cytotoxic activities of brevinin1 peptides and their synthetic analogs (amidated C‐terminus) were investigated and compared. All the peptides except the acidic forms showed antibacterial activity against all tested Gram‐positive and Gram‐negative bacteria. They exhibited low hemolysis on human erythrocytes and showed potent cytotoxic activity against Hep 3B cancer cell line. Upon amidation, the peptides showed increased activity against the tested microbes without altering their hemolytic and cytotoxic properties. The study also emphasizes the need for screening endemic amphibian fauna of Western Ghats, as a potential source of host defense peptides with possible therapeutic applications in the future.     

Low-molecular-mass peptides from the venom of the Amazonian viper Bothrops atrox protect against brain mitochondrial swelling in rat: Potential for neuroprotection

The neurodegenerative diseases are important causes of morbidity and mortality in Western countries. Common mechanisms of toxicity involving mitochondrial damage have been suggested; however, a definitive treatment has not yet been found. Therefore, there has been great interest in the development of mitochondria-targeted protective compounds for the treatment of neuropathies. Animal toxins represent a promising source of new molecules with neuroprotective activity and potential to originate new drugs. We present here the effects of a low-molecular-mass peptides fraction (Ba-V) from Bothrops atrox snake venom, on rat brain mitochondrial function. Ba-V did not induce the mitochondrial swelling and moreover, was as effective as cyclosporin A (CsA) to inhibit the calcium/phosphate-induced swelling, which indicates its potential to prevent the mitochondrial permeability transition (MPT). The membrane electrochemical potential, the oxygen consumption during states-3 and -4 respirations as well as the respiratory control ratio (RCR) were not affected by Ba-V. Additionally, Ba-V did not induce reactive oxygen species (ROS) generation. Interestingly, Ba-V did not protect against the generation of ROS induced by t-BOH, which suggests a protection mechanism other than ROS scavenging. Given the important role of the mitochondrial damage and, more specifically, of MPT, in the development of neuropathies, Ba-V might be useful in the future strategies for the treatment of these diseases.     

De novo sequencing of peptides from the parotid secretion of the cane toad, Bufo marinus (Rhinella marina)

Amphibian skin secretions are well known as a rich source of bioactive peptides. However, little is known about the presence or role of peptides in the highly toxic, parotid secretion of the cane toad or giant toad, Bufo marinus (Rhinella marina), though small molecule bufadienolides, which act as potent cardiotoxins, have been described. In the current study we used RP-HPLC, MALDI-TOF mass spectrometry and tandem mass spectrometry to analyze and determine the first sequences of peptides from the parotid secretion of B. marinus. We show that peptides in the range of 900-2500 Da are indeed present, however in extremely low abundance. Despite the low abundance, the sequences of 14 peptides were determined, several of which match fragments of larger cellular proteins, yet none share substantial homology with defensive or anti-microbial peptides reported from frog skin secretions. We conclude that peptides are present in the parotid glands of B. marinus only in very low quantities and that they are likely to be breakdown products of proteins involved in cell maintenance. Given these results, we conclude that peptides are unlikely to contribute directly to the high toxicity of the cane toad.     

Di-(2-ethylhexyl) phthalate enhances cytokine release from group 2 innate lymphoid cells in the presence of interleukin-33

Epidemiological and experimental studies have shown that di-(2-ethylhexyl) phthalate (DEHP), a plasticizer, can aggravate allergic diseases. DEHP promotes adaptive immune responses, although its effect on the innate immune system remains largely unknown. The present study investigated the effects of DEHP on group 2 innate lymphoid cells (ILC2) that produce Th2 cytokines in response to epithelial cell-derived cytokines, such as interleukin (IL)-33. ILC2 (lineage-negative, CD45.2⁺, Sca1⁺, KLRG1⁺) were isolated from the lungs of C57BL/6 J mice. Co-exposure to DEHP and IL-33 significantly increased IL-5 release from ILC2, whose level was higher than that of the vehicle and IL-33 alone. The effects of DEHP in the presence of IL-33 showed an inverted-U dose-response. The present is the first report showing that DEHP exacerbates allergy through the innate immune system.     

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC₅₀ values of 49 μg/ml and 30 μg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21^cip1 and p27^kip1) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature.     

Identification and characterisation of a novel antimicrobial polypeptide from the skin secretion of a Chinese frog (Rana chensinensis)

Amphibians secrete small antimicrobial polypeptides from their skin that have been explored as alternatives to conventional antibiotics. In this study, mass spectrometry was used to identify and characterise protein secretions from the skin of a Chinese frog, Rana chensinensis. The skin of this kind of frog has been used in traditional Chinese medicine for centuries as a remedy against inflammation. A novel antimicrobial peptide was identified and the characteristics of this peptide were analysed using far-ultraviolet circular dichroism. When dissolved in aqueous solution, the peptide displayed a high level of random coil structure, in contrast to a more ordered α-helical structure when dissolved in 50% trifluoroethanol. Functional studies showed that this peptide has potent antimicrobial activity both against Gram-positive and Gram-negative bacteria and has extremely low haemolytic activity to human red blood cells. Taken together, these studies suggest that this novel peptide can be further developed as an antimicrobial agent.     

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K⁺ (K_ATP) channels, increasing intracellular Ca²⁺ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K_ATP channel opener and 10 μM nifedipine, a Ca²⁺ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K_ATP and L-type Ca²⁺ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling.     

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra)

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name ‘najanalgesin’. The LD₅₀ of the crude venom and najanalgesin were 0.89 mg/kg and 2.69 mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445 mg/kg) in a dose-dependent manner. Najanalgesin (1 mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445 mg/kg and remained for 6 h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3 h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0 mg/kg, respectively. Pretreatment with atropine (1 mg/kg) or naloxone (3 mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.     

Peptides with differential cytolytic activity from skin secretions of the lemur leaf frog Hylomantis lemur (Hylidae: Phyllomedusinae).

Two peptides with differential cytolytic activity against bacteria, a fungus pathogenic to amphibians, and mammalian cells were isolated from norepinephrine-stimulated skin secretions of the Lemur leaf frog Hylomantis lemur Boulenger, 1882. Dermaseptin-L1 (GLWSKIKEAAKAAGKAALNAVTGLVNQGDQPS) was active against the Gram-negative bacterium Escherichia coli (MIC=8 microM) but inactive against the Gram-positive bacterium Staphylococcus aureus. This peptide inhibited growth of zoospores of the chytrid fungus Batrachochytrium dendrobatidis at concentrations above 25 microM but did not completely inhibit growth at 100 microM. Phylloseptin-L1 (LLGMIPLAISAISALSKL.NH2) was active against S. aureus (MIC=8 microM) but was inactive against E. coli. This peptide also inhibited growth of B. dendrobatidis zoospores at concentrations above 25 microM with complete inhibition at 100 microM. Dermaseptin-L1 showed selective cytolytic activity against HepG2 human hepatoma-derived cells (LC50=45 microM) compared with human erythrocytes (LC50=200 microM) whereas phylloseptin-L1 was approximately equipotent against both HepG2 cells (LC50=35 microM) and erythrocytes (LC50=40 microM).     

Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi)

Two thrombin-like enzymes were purified from the venom of Agkistrodon caliginosus. Both were homogeneous on polyacrylamide gel electrophoresis, hydrolyzed $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester and did not show any caseinolytic activity. The molecular weights of the enzymes were estimated to be about 34,000–37,000 and 42,000–43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel-filtration on Sephadex G-100 column. One enzyme was purified from the venom by gel-filtration on Sephadex G-100 column, CM-Sephadex C-50 and DEAE-Sephadex A-50 chromatographies, affinity chromatography on p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B and gel-filtration on Sephadex G-100 column. The second enzyme was separated from the first enzyme by the DEAE-Sephadex A-50 chromatography mentioned in the above procedure and was isolated by affinity chromatography on arginine-Sepharose 4B and gel-filtration on Sephadex G-100 column. From 4 g of the venom, 0.88 mg of the first enzyme and 1.18 mg of the second enzyme were obtained, with specific activities of 118 and 139 NIH units per mg of protein, respectively.     

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p < 0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flow-cytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity.     

Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin

The ability of crude venom and a basic phospholipase A₂ (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H₁ antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor l-NAME (100 nmol/site), tachykinin NK₁ antagonist SR140333 (1 nmol/site) and bradykinin B₂ receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.     

Pharmacological studies with scorpion (Palamneus gravimanus) venom: evidence for the presence of histamine

The venom from the scorpion Palamneus gravimanus caused, in cats, a pronounced hypotensive effect which was blocked by mepyramine. The hypotensive effect was observed in spinal and reserpinized cats, was not affected by carotid sinus and body denervation and bilateral vagotomy, and was not blocked by hexamethonium and atropine treatment. The venom produced hypertension in guinea pigs and rats which was blocked by tolazoline and in the case of the guinea pigs by mepyramine and by phenoxybenzamine in the rats. Treatment of rats with compound 48/80 did not prevent the hypertensive effect of the venom, showing that it is probably due to histamine or histamine-like substance in the venom and not due to releasing of histamine. Dialysis greatly attenuated the hypotensive effect and prevented the hypertensive action of the venom. Histamine in the venom was identified by paper chromatography, and biological assays showed a histamine-like activity corresponding to 5–7.2 μg histamine per mg dry venom. Both the crude and the dialysed venoms produced positive inotropic and chronotropic effects on isolated rabbit's heart that were not affected by pyribenzamine but were blocked by propranolol. The cardiac stimulant effect of the venom was absent in reserpinized hearts. It is concluded that histamine or histamine-like substance is present in P. gravimanus venom; however, all of the venom effects are not due to the presence of that substance.     

Purification of a myotoxin from the toadfish Thalassophryne maculosa (Günter) venom

Venom was milked by gently pressing the base of the opercular and dorsal fin spines. Three fractions were obtained by molecular exclusion high pressure liquid chromatography (HPLC) (Protein Pak™ 125SW, Millipore Corporation) column, but only the last one with 22.7 min retention time (rt) was biological active (TmPP-22.7). This fraction was rechromatographed on reversed phase HPLC chlorobutylsilane columns (C4, Vydac) nine fractions were obtained, but only one (TmC4-47.2) with 47.2 min rt was biologically active. MALD-TOF mass analysis was carried out on two samples of TmC-47.2 and the results were 15,161.36 and 15,154.70 a.m.u., respectively. Raw venom (1040 μg/ml) depolarised frog (Hyla crepitans) muscle irreversibly from −85 (−88, −81) mV (n=20, median and its 95% CI) to −18 (−24, −15) mV (n=24). The biological activity in TmPP-22.7 (38 μg/ml), which depolarised muscle fibres from −79 (−82, −76) mV (n=20) to −63 (−69 −57) mV (n=24). The depolarising fraction was TmC4-47.2 (50 μg/ml) which depolarised muscles from −87 (91, −82) mV (n=33) to −63 (−76 −51) mV (n=53); the depolarising effect at this concentration was completely reversed on washing with normal saline for 2 h. Muscles treated with 1 μM tetrodotoxin (TTX) were depolarised from −80 (−85, −72) mV (n=49) to −44 (−56, −31) mV (n=44) when 100 μg/ml TmC4-47.2 were applied with TTX; washing 130 min with 1 μM TTX repolarised to −59 (−69, −50) mV (n=25). We also present evidence that TmC4-47.2 induces myonecrosis in mice.     

Histopathological changes in mouse and rat skin injected with venom sac extracts of the oriental hornet (Vespa orientalis)

ICR white and C57 black mice and Charles Rivers rats were injected intradermally with a dialysed extract of venom sac of the Oriental hornet. Changes were observed throughout the skin. The epidermis underwent hyperplasia and hairless patches and depigmentation were observed in black mice. Acute inflammation spread throughout the dermis, evolving into a chronic form. The muscle fibers showed degeneration. The extent of the changes was dependent upon the species involved: the C57 black mice were the most affected and the rats the least.     

Amino acid sequence of a kinin-releasing enzyme, KR-E-1, from the venom of Agkistrodon caliginosus (Kankoku-mamushi)

The amino acid sequence of a bradykinin-releasing enzyme, named KR-E-1, isolated from the venom of Agkistrodon caliginosus (Kankoku-mamushi) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, hydroxylamine, achromobacter protease I, trypsin, V8 protease, arginine endopeptidase, and endoproteinase Asp-N. KR-E-1 consisted of 235 amino acids and showed conservation of the catalytic amino acid residues (His⁵⁷, Asp¹⁰², and Ser¹⁹⁵) of the chymotrypsin family of serine protease in its amino acid sequence. The carboxy-terminal amino acid, Phe, was determined using carboxypeptidase Y. This enzyme contains glucosamine and an N-linked glycosylation site. KR-E-1 showed 32, 31, 65, 65, and 67% sequence homology to human kallikrein, bovine thrombin, KN-BJ 2, elegaxobin, and elegaxobin II, respectively. The characteristic of structure of KR-E-1 was found to involve hydrophobic amino acid residues abundantly localizing in positions 1-50, with lysine residues abundantly localizing in positions 73-101.