Ultrafast mode-locked laser in nanophotonic lithium niobate.

Mode-locked lasers (MLLs) generate ultrashort pulses with peak powers substantially exceeding their average powers. However, integrated MLLs that drive ultrafast nanophotonic circuits have remained elusive because of their typically low peak powers, lack of controllability, and challenges when integrating with nanophotonic platforms. In this work, we demonstrate an electrically pumped actively MLL in nanophotonic lithium niobate based on its hybrid integration with a III-V semiconductor optical amplifier. Our MLL generates [Formula: see text]4.8-ps optical pulses around 1065 nm at a repetition rate of ∼10 GHz, with energies exceeding 2.6 pJ and peak powers beyond 0.5 W. The repetition rate and the carrier-envelope offset frequency of the output can be controlled in a wide range by using the driving frequency and the pump current, providing a route for fully stabilized on-chip frequency combs.

Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors.

The cell and gene therapy industry has employed the same plasmid technology for decades in vaccination, cell and gene therapy, and as a raw material in viral vector and RNA production. While canonical plasmids contain antibiotic resistance markers in bacterial backbones greater than 2,000 base pairs, smaller backbones increase expression level and durability and reduce the cell-transfection-associated toxicity and transgene silencing that can occur with canonical plasmids. Therefore, the small backbone and antibiotic-free selection method of Nanoplasmid vectors have proven to be a transformative replacement in a wide variety of applications, offering a greater safety profile and efficiency than traditional plasmids. This review provides an overview of the Nanoplasmid technology and highlights its specific benefits for various applications with examples from recent publications.

Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

Sexual behavior shapes male genitourinary microbiome composition.

The origin, composition, and significance of the distal male urethral microbiome are unclear, but vaginal microbiome dysbiosis is linked to new sex partners and several urogynecological syndromes. We characterized 110 urethral specimens from men without urethral symptoms, infections, or inflammation using shotgun metagenomics. Most urethral specimens contain characteristic lactic acid bacteria and Corynebacterium spp. In contrast, several bacteria associated with vaginal dysbiosis were present only in specimens from men who reported vaginal intercourse. Sexual behavior, but not other evaluated behavioral, demographic, or clinical variables, strongly associated with inter-specimen variance in urethral microbiome composition. Thus, the male urethra supports a simple core microbiome that is established independent of sexual exposures but can be re-shaped by vaginal sex. Overall, the results suggest that urogenital microbiology and sexual behavior are inexorably intertwined, and show that the male urethra harbors female urogenital pathobionts.

Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection.

Hepatic gene therapy by delivering non-integrating therapeutic vectors in newborns remains challenging due to the risk of dilution and loss of efficacy in the growing liver. Previously we reported on hepatocyte transfection in piglets by intraportal injection of naked DNA vectors. Here, we established delivery of naked DNA vectors to target periportal hepatocytes in weaned pigs by hydrodynamic retrograde intrabiliary injection (HRII). The surgical procedure involved laparotomy and transient isolation of the liver. For vector delivery, a catheter was placed within the common bile duct by enterotomy. Under optimal conditions, no histological abnormalities were observed in liver tissue upon pressurized injections. The transfection of hepatocytes in all tested liver samples was observed with vectors expressing luciferase from a liver-specific promoter. However, vector copy number and luciferase expression were low compared to hydrodynamic intraportal injection. A 10-fold higher number of vector genomes and luciferase expression was observed in pigs using a non-integrating naked DNA vector with the potential for replication. In summary, the HRII application was less efficient (i.e., lower luciferase activity and vector copy numbers) than the intraportal delivery method but was significantly less distressful for the piglets and has the potential for injection (or re-injection) of vector DNA by endoscopic retrograde cholangiopancreatography.

Evaluation of Clinical, Gram Stain, and Microbiological Cure Outcomes in Men Receiving Azithromycin for Acute Nongonococcal Urethritis: Discordant Cures Are Associated With Mycoplasma genitalium Infection.

BACKGROUND: In men with nongonococcal urethritis (NGU), clinicians and patients rely on clinical cure to guide the need for additional testing/treatment and when to resume sex, respectively; however, discordant clinical and microbiological cure outcomes do occur. How accurately clinical cure reflects microbiological cure in specific sexually transmitted infections (STIs) is unclear. METHODS: Men with NGU were tested for Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis, urethrotropic Neisseria meningitidis ST-11 clade strains, and Ureaplasma urealyticum (UU). Men received azithromycin 1 g and returned for a 1-month test-of-cure visit. In MG infections, we evaluated for the presence of macrolide resistance-mediating mutations (MRMs) and investigated alternate hypotheses for microbiological treatment failure using in situ shotgun metagenomic sequencing, phylogenetic analysis, multilocus sequence typing analyses, and quantitative PCR. RESULTS: Of 280 men with NGU, 121 were included in this analysis. In the monoinfection group, 52 had CT, 16 had MG, 7 had UU, 10 had mixed infection, and 36 men had idiopathic NGU. Clinical cure rates were 85% for CT, 100% for UU, 50% for MG, and 67% for idiopathic NGU. Clinical cure accurately predicted microbiological cure for all STIs, except MG. Discordant results were significantly associated with MG-NGU and predominantly reflected microbiological failure in men with clinical cure. Mycoplasma genitalium MRMs, but not MG load or strain, were strongly associated with microbiological failure. CONCLUSIONS: In azithromycin-treated NGU, clinical cure predicts microbiological cure for all STIs, except MG. Nongonococcal urethritis management should include MG testing and confirmation of microbiological cure in azithromycin-treated MG-NGU when MRM testing is unavailable.

Improved Potency and Safety of DNA-Encoded Antibody Therapeutics Through Plasmid Backbone and Expression Cassette Engineering.

DNA-encoded delivery of antibodies presents a labor- and cost-effective alternative to conventional antibody therapeutics. This study aims to improve the potency and safety of this approach by evaluating various plasmid backbones and expression cassettes. In vitro, antibody levels consistently improved with decreasing sizes of backbone, ranging from conventional to minimal. In vivo, following intramuscular electrotransfer in mice, the correlation was less consistent. While the largest conventional plasmid (10.2 kb) gave the lowest monoclonal antibody (mAb) levels, a regular conventional plasmid (8.6 kb) demonstrated similar levels as a minimal Nanoplasmid (6.8 kb). A reduction in size beyond a standard conventional backbone thus did not improve mAb levels in vivo. Cassette modifications, such as swapping antibody chain order or use of two versus a single encoding plasmid, significantly increased antibody expression in vitro, but failed to translate in vivo. Conversely, a significant improvement in vivo but not in vitro was found with a set of muscle-specific promoters, of which a newly engineered variant gave roughly 1.5- to 2-fold higher plasma antibody concentrations than the ubiquitous CAG promoter. In conclusion, despite the limited translation between in vitro and in vivo, we identified various clinically relevant improvements to our DNA-based antibody platform, both in potency and biosafety.

Identification of KMT2A-ARHGEF12 fusion in a child with a high-grade B-cell lymphoma.

Rearrangements involving KMT2A are common in de novo and therapy-related acute myeloid and lymphoblastic leukemias. There is a diverse recombinome associated with KMT2A involving at least 135 partner genes, with more being discovered due to advances in molecular genetic diagnostics. KMT2A-ARHGEF12 fusion has only rarely been reported, in five cases of acute leukemia and a single case of high-grade B-cell lymphoma. We present a 12-year-old boy with high-grade B-cell lymphoma and KMT2A-ARHGEF12 fusion, whose clinical, morphologic, phenotypic and genotypic profile is strikingly similar to the other case of high grade B cell lymphoma, both otherwise perfectly mimicking Burkitt lymphoma.

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies.

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of broadly active ADCC antibodies may require mutations that enable high-affinity antigen recognition along with mutations that optimize factors contributing to functional ADCC activity.

Nearly four decades after the human immunodeficiency virus (HIV for short) was first identified, the search for a vaccine still continues. An effective immunisation would require elements that coax the human immune system into making HIV-specific antibodies ­ the proteins that can recognise, bind to and deactivate the virus. Crucially, antibodies can also help white blood cells to target and destroy cells infected with HIV. This 'antibody-dependent cellular cytotoxicity' could be a key element of a successful vaccine, yet it has received less attention than the ability for antibodies to directly neutralize the virus. In particular, it is still unclear how antibodies develop the ability to flag HIV-infected cells for killing. Indeed, over the course of an HIV infection, an immune cell goes through genetic changes that tweak the 3D structure of the antibodies it manufactures. This process can improve the antibodies' ability to fight off the virus, but it was still unclear how it would shape antibody-dependent cellular cytotoxicity. To investigate this question, Doepker et al. retraced how the genes coding for six antibody families changed over time in an HIV-carrying individual. This revealed that antibodies could not initially trigger antibody-dependent cellular cytotoxicity. The property emerged and improved thanks to two types of alterations in the genetic sequences. One set of changes increased how tightly the antibodies could bind to the virus, targeting sections of the antibodies that can often vary. The second set likely altered the 3D structure in others ways, potentially affecting how antibodies bind the virus or how they interact with components of the immune system that help to kill HIV-infected cells. These alterations took place in segments of the antibodies that undergo less change over time. Ultimately, the findings by Doepker et al. suggest that an efficient HIV vaccine may rely on helping antibodies to evolve so they can bind more tightly to the virus and trigger cellular cytotoxicity more strongly.

Analytical and Clinical Sample Performance Characteristics of the Onclarity Assay for the Detection of Human Papillomavirus.

The objective of this study was to determine the result reproducibility and performance of the BD Onclarity human papillomavirus (HPV) assay (Onclarity) on the BD Viper LT platform using both contrived and clinical specimens. Reproducibility was assessed in BD SurePath liquid-based cytology (LBC) medium (SurePath) using contrived panels (HPV genotype 16 [HPV16] positive, HPV18 positive, or HPV45 positive) or clinical specimens (HPV16, -18, -31, -33/58, -45, or -52 positive or HPV negative). In addition, specimens from 3,879 individuals from the Onclarity trial were aliquoted prior to or following cytology processing and tested for HPV. Finally, specimens were collected using either the Cervex-Brush or Cytobrush (or Cytobrush/spatula) for comparison of HPV results. Contrived specimens showed >95% concordance with the expected results, and pooled clinical specimens had standard deviations and coefficients of variation ranging from 0.87 to 1.86 and 2.9% to 5.6%, respectively. For precytology and postcytology aliquot analyses, specimens showed >98.0% overall agreement and mean differences in cycle threshold (CT ) scores for HPV ranging from -0.07 to 0.31. Positivity rates were close between the Cervex-Brush and Cytobrush/spatula for all age groups tested. Onclarity results are reproducible and reliable, regardless of sample collection before or after cytology aliquoting. Onclarity performs well regardless of the method of specimen collection (Cervex-Brush or Cytobrush/spatula) for cervical cancer screening.

Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models.

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

Nanoplasmid Vectors Co-expressing Innate Immune Agonists Enhance DNA Vaccines for Venezuelan Equine Encephalitis Virus and Ebola Virus.

DNA vaccines expressing codon-optimized Venezuelan equine encephalitis virus (VEEV) and Ebola virus (EBOV) glycoprotein genes provide protective immunity to mice and nonhuman primates when delivered by intramuscular (IM) electroporation (EP). To achieve equivalent protective efficacy in the absence of EP, we evaluated VEEV and EBOV DNA vaccines constructed using minimalized Nanoplasmid expression vectors that are smaller than conventional plasmids used for DNA vaccination. These vectors may also be designed to co-express type I interferon inducing innate immune agonist genes that have an adjuvant effect. Nanoplasmid vaccinated mice had increased antibody responses as compared to those receiving our conventional pWRG7077-based vaccines when delivered by IM injection, and these responses were further enhanced by the inclusion of the innate immune agonist genes. The Nanoplasmid VEEV DNA vaccines also significantly increased protection against aerosol VEEV challenge as compared to the pWRG7077 VEEV DNA vaccine. Although all mice receiving the pWRG7077 and Nanoplasmid EBOV DNA vaccines at the dose tested survived EBOV challenge, only mice receiving the Nanoplasmid EBOV DNA vaccine that co-expresses the innate immune agonist genes failed to lose weight after challenge. Our results suggest that Nanoplasmid vectors can improve the immunogenicity and protective efficacy of alphavirus and filovirus DNA vaccines.

No Pathogen-Specific Sign or Symptom Predicts the Etiology of Monomicrobial Nongonococcal Urethritis in Men.

Identifying pathogen-specific signs or symptoms of nongonococcal urethritis could improve syndromic management accuracy. We evaluated nongonococcal urethritis signs and symptoms in 220 men with single-pathogen infections (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, or Ureaplasma urealyticum) or idiopathic urethritis. No individual sign or symptom accurately predicted the infectious etiology.

Development of a SimpleProbe real-Time PCR Assay for rapid detection and identification of the US novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC).

Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.

Aetiology and prevalence of mixed-infections and mono-infections in non-gonococcal urethritis in men: a case-control study.

OBJECTIVES: Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) cause the majority of non-gonococcal urethritis (NGU). The role of Ureaplasma urealyticum (UU) in NGU is unclear. Prior case-control studies that examined the association of UU and NGU may have been confounded by mixed infections and less stringent criteria for controls. The objective of this case-control study was to determine the prevalence and aetiology of mixed infections in men and assess if UU monoinfection is associated with NGU. METHODS: We identified 155 men with NGU and 103 controls. Behavioural and clinical information was obtained and men were tested for Neisseria gonorrhoeae and CT, MG, UU and Trichomonas vaginalis (TV). Men who were five-pathogen negative were classified as idiopathic urethritis (IU). RESULTS: Twelve per cent of NGU cases in which a pathogen was identified had mixed infections, mostly UU coinfections with MG or CT; 27% had IU. In monoinfected NGU cases, 34% had CT, 17% had MG, 11% had UU and 2% had TV. In controls, pathogens were rarely identified, except for UU, which was present in 20%. Comparing cases and controls, NGU was associated with CT and MG monoinfections and mixed infections. UU monoinfection was not associated with NGU and was almost twice as prevalent in controls. Men in both the case and control groups who were younger and who reported no prior NGU diagnosis were more likely to have UU (OR 0.97 per year of age, 95% CI 0.94 to 0.998 and OR 6.3, 95% CI 1.4 to 28.5, respectively). CONCLUSIONS: Mixed infections are common in men with NGU and most of these are UU coinfections with other pathogens that are well-established causes of NGU. UU monoinfections are not associated with NGU and are common in younger men and men who have never previously had NGU. Almost half of NGU cases are idiopathic.

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors.

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

Detection of Rectal Chlamydia trachomatis in Heterosexual Men Who Report Cunnilingus.

BACKGROUND: Rectal infection with Chlamydia trachomatis (CT) is frequent in women who deny receptive anal sex and is thought to arise from autoinoculation of the rectum from vaginal secretions. An alternate hypothesis is that oral sex inoculates and establishes gastrointestinal tract infection. Distinguishing these hypotheses is difficult in women. In men, autoinoculation is unlikely and heterosexual men frequently perform oral sex, but rarely participate in receptive anal exposure behaviors. METHODS: We enrolled high-risk men with and without nongonococcal urethritis who presented to a sexually transmitted infection clinic in Indianapolis, Indiana. Urine and rectal swabs were collected and tested for urogenital and rectal CT, Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG). Men completed surveys concerning symptoms, sexual orientation, and detailed recent and lifetime oral and anal sexual behaviors. RESULTS: Rectal CT was detected in 2/84 (2.4%) heterosexual men who reported cunnilingus, but no lifetime receptive anal behaviors. All of the men who denied receptive anal behaviors were negative for rectal NG and MG. In homosexual and bisexual men, rectal CT prevalence was high (9.7%), and rectal NG (4.8%) and MG (4.8%) were also detected. CONCLUSIONS: We detected rectal CT infections in heterosexual men who reported cunnilingus but denied receptive anal behaviors. Oral sex may be a risk factor for rectal CT infection via oral inoculation of the gastrointestinal tract.

Kappa chain maturation helps drive rapid development of an infant HIV-1 broadly neutralizing antibody lineage.

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.