Metaproteomic portrait of the healthy human gut microbiota.

Gut metaproteomics can provide direct evidence of microbial functions actively expressed in the colonic environments, contributing to clarify the role of the gut microbiota in human physiology. In this study, we re-analyzed 10 fecal metaproteomics datasets of healthy individuals from different continents and countries, with the aim of identifying stable and variable gut microbial functions and defining the contribution of specific bacterial taxa to the main metabolic pathways. The "core" metaproteome included 182 microbial functions and 83 pathways that were identified in all individuals analyzed. Several enzymes involved in glucose and pyruvate metabolism, along with glutamate dehydrogenase, acetate kinase, elongation factors G and Tu and DnaK, were the proteins with the lowest abundance variability in the cohorts under study. On the contrary, proteins involved in chemotaxis, response to stress and cell adhesion were among the most variable functions. Random-effect meta-analysis of correlation trends between taxa, functions and pathways revealed key ecological and molecular associations within the gut microbiota. The contribution of specific bacterial taxa to the main biological processes was also investigated, finding that Faecalibacterium is the most stable genus and the top contributor to anti-inflammatory butyrate production in the healthy gut microbiota. Active production of other mucosal immunomodulators facilitating host tolerance was observed, including Roseburia flagellin and lipopolysaccharide biosynthetic enzymes expressed by members of Bacteroidota. Our study provides a detailed picture of the healthy human gut microbiota, contributing to unveil its functional mechanisms and its relationship with nutrition, immunity, and environmental stressors.

Benchmarking low- and high-throughput protein cleanup and digestion methods for human fecal metaproteomics.

The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although high-throughput protein cleanup and digestion methods are increasingly used in shotgun proteomics, no studies have yet critically compared such protocols using human fecal samples. In this study, human fecal protein extracts were processed using several different protocols based on three main approaches: filter-aided sample preparation (FASP), solid-phase-enhanced sample preparation (SP3), and suspension trapping (S-Trap). These protocols were applied in both low-throughput (i.e., microtube-based) and high-throughput (i.e., microplate-based) formats, and the final peptide mixtures were analyzed by liquid chromatography coupled to high-resolution tandem mass spectrometry. The FASP-based methods and the combination of SP3 with in-StageTips (iST) yielded the best results in terms of the number of peptides identified through a database search against gut microbiome and human sequences. The efficiency of protein digestion, the ability to preserve hydrophobic peptides and high molecular weight proteins, and the reproducibility of the methods were also evaluated for the different protocols. Other relevant variables, including interindividual variability of stool, duration of protocols, and total costs, were considered and discussed. In conclusion, the data presented here can significantly contribute to the optimization and standardization of sample preparation protocols in human fecal metaproteomics. Furthermore, the promising results obtained with the high-throughput methods are expected to encourage the development of automated workflows and their application to large-scale gut microbiome studies.IMPORTANCEFecal metaproteomics is an experimental approach that allows the investigation of gut microbial functions, which are involved in many different physiological and pathological processes. Standardization and automation of sample preparation protocols in fecal metaproteomics are essential for its application in large-scale studies. Here, we comparatively evaluated different methods, available also in a high-throughput format, enabling two key steps of the metaproteomics analytical workflow (namely, protein cleanup and digestion). The results of our study provide critical information that may be useful for the optimization of metaproteomics experimental pipelines and their implementation in laboratory automation systems.

An outbreak of COVID-19 after a pilgrimage to Medjugorje due to Delta sub-lineages.

INTRODUCTION: A COVID-19 outbreak occurred at the end of October 2021 among pilgrims returning from Medjugorje (Bosnia and Herzegovina). METHODOLOGY: Whole genome sequencing (WGS) of SARS-CoV-2, epidemiological data, and phylogenetic analysis were used to reconstruct outbreak dynamics. RESULTS: The results suggest that only in one case, associated with the SARS-CoV-2 sub-lineage AY.9.2, it is possible to trace back the place of contagion to Medjugorje, while the other cases were likely to be acquired in the country of origin. CONCLUSIONS: The combined use of phylogenetic data derived from WGS, and epidemiological data allowed us to study epidemic dynamics and to formulate a possible hypothesis on the place of exposure to SARS-CoV-2. The identification of different sub-lineages of the SARS-CoV-2 Delta variant also suggested that different chains of transmission contributed to the outbreak.

Metaproteomic assessment of gut microbial and host functional perturbations in Helicobacter pylori-infected patients subjected to an antimicrobial protocol.

The impact of therapeutic interventions on the human gut microbiota (GM) is a clinical issue of paramount interest given the strong interconnection between microbial dynamics and human health. Orally administered antibiotics are known to reduce GM biomass and modify GM taxonomic profile. However, the impact of antimicrobial therapies on GM functions and biochemical pathways has scarcely been studied. Here, we characterized the fecal metaproteome of 10 Helicobacter pylori-infected patients before (T0) and after 10 days (T1) of a successful quadruple therapy (bismuth, tetracycline, metronidazole, and rabeprazole) and 30 days after therapy cessation (T2), to investigate how GM and host functions change during the eradication and healing processes. At T1, the abundance ratio between microbial and host proteins was reversed compared with that at T0 and T2. Several pathobionts (including Klebsiella, Proteus, Enterococcus, Muribaculum, and Enterocloster) were increased at T1. Therapy reshaped the relative contributions of the functions required to produce acetate, propionate, and butyrate. Proteins related to the uptake and processing of complex glycans were increased. Microbial cross-feeding with sialic acid, fucose, and rhamnose was enhanced, whereas hydrogen sulfide production was reduced. Finally, microbial proteins involved in antibiotic resistance and inflammation were more abundant after therapy. Moreover, a reduction in host proteins with known roles in inflammation and H. pylori-mediated carcinogenesis was observed. In conclusion, our results support the use of metaproteomics to monitor drug-induced remodeling of GM and host functions, opening the way for investigating new antimicrobial therapies aimed at preserving gut environmental homeostasis.

Vaccination and Antiviral Treatment Reduce the Time to Negative SARS-CoV-2 Swab: A Real-Life Study.

Clinical trials demonstrated the role of vaccines and antiviral treatments against SARS-CoV-2 in reducing the likelihood of disease progression and death. However, there are limited data available regarding the time to negativity of people who received these treatments. Further, several comorbidities and risk factors might affect the impact of vaccines and antiviral treatments. To this end, we aimed to evaluate and disentangle the impact of anti-SARS-CoV-2 treatments and that of underlying clinical factors associated with a shortened length of SARS-CoV-2 infection. Hence, we recorded the timeframe of positive nasopharyngeal swab in people infected while being hospitalized for reasons other than SARS-CoV-2 infection. All patients who died or were discharged with a positive swab were excluded from the study. A total of 175 patients were included in this study. Clinical conditions encompass malignancies, immunological disorders, cardiovascular, metabolic, neurodegenerative, and chronic kidney disease. Most of the participants (91.4%) were vaccinated before admission to the hospital, and 65.1% received antiviral treatment within three days after the symptom's onset. Unvaccinated patients had a longer median time to negativity than people who received at least two doses of vaccine (18 vs. 10 days). Concerning the clinical conditions of all patients, multivariate analysis highlighted a lower probability of 14-day conversion of antigenic test positivity in patients with hematological malignancy, including those vaccinated and those exposed to antiviral therapies. In conclusion, our data showed that prompt administration of antiviral treatments accelerates the clearance of SARS-CoV-2. Further, in the elderly patients under study, previous vaccination and antiviral treatment synergize to reduce time to negativity. This translates into a shorter hospitalization time and a lower risk of transmission through patients and connected healthcare workers in a hospital ward setting, with considerable improvement in cost-effective care management.

Meta4P: A User-Friendly Tool to Parse Label-Free Quantitative Metaproteomic Data and Taxonomic/Functional Annotations.

We present Meta4P (MetaProteins-Peptides-PSMs Parser), an easy-to-use bioinformatic application designed to integrate label-free quantitative metaproteomic data with taxonomic and functional annotations. Meta4P can retrieve, filter, and process identification and quantification data from three levels of inputs (proteins, peptides, PSMs) in different file formats. Abundance data can be combined with taxonomic and functional information and aggregated at different and customizable levels, including taxon-specific functions and pathways. Meta4P output tables, available in various formats, are ready to be used as inputs for downstream statistical analyses. This user-friendly tool is expected to provide a useful contribution to the field of metaproteomic data analysis, helping make it more manageable and straightforward.

Cross-sectional analysis of the humoral response after SARS-CoV-2 vaccination in Sardinian multiple sclerosis patients, a follow-up study.

Monitoring immune responses to SARS-CoV-2 vaccination and its clinical efficacy over time in Multiple Sclerosis (MS) patients treated with disease-modifying therapies (DMTs) help to establish the optimal strategies to ensure adequate COVID-19 protection without compromising disease control offered by DMTs. Following our previous observations on the humoral response one month after two doses of BNT162b2 vaccine (T1) in MS patients differently treated, here we present a cross-sectional and longitudinal follow-up analysis six months following vaccination (T2, n=662) and one month following the first booster (T3, n=185). Consistent with results at T1, humoral responses were decreased in MS patients treated with fingolimod and anti-CD20 therapies compared with untreated patients also at the time points considered here (T2 and T3). Interestingly, a strong upregulation one month after the booster was observed in patients under every DMTs analyzed, including those treated with fingolimod and anti-CD20 therapies. Although patients taking these latter therapies had a higher rate of COVID-19 infection five months after the first booster, only mild symptoms that did not require hospitalization were reported for all the DMTs analyzed here. Based on these findings we anticipate that additional vaccine booster shots will likely further improve immune responses and COVID-19 protection in MS patients treated with any DMT.

Metagenomic Changes of Gut Microbiota following Treatment of Helicobacter pylori Infection with a Simplified Low-Dose Quadruple Therapy with Bismuth or Lactobacillus reuteri.

BACKGROUND: Probiotic supplementation to antibiotic regimens against Helicobacter pylori infection has been proposed to improve eradication rate and to decrease detrimental effects on gut microbiota. AIMS: To evaluate microbiota modifications due to a low-dose quadruple therapy with bismuth or Lactobacillus reuteri. METHODS: Forty-six patients infected with H. pylori were prospectively enrolled in a single-centre, randomized controlled trial to receive b.i.d. with meals for 10 days low-dose quadruple therapy consisting of rabeprazole 20 mg and bismuth (two capsules of Pylera® plus 250 mg each of tetracycline and metronidazole), or the same dose of rabeprazole and antibiotics plus Gastrus® (L. reuteri), one tablet twice-a-day for 27 days. Stool samples were collected at the enrolment, at the end and 30-40 days after the treatment. Gut microbiota composition was investigated with 16S rRNA gene sequencing. RESULTS: Eradication rate was by ITT 78% in both groups, and by PP analysis 85.7% and 95.5% for Gastrus® and bismuth group, respectively. Alpha and beta diversity decreased at the end of treatment and was associated with a reduction of bacterial genera beneficial for gut homeostasis, which was rescued 30-40 days later in both groups, suggesting a similar impact of the two regimens in challenging bacterial community complexity. CONCLUSIONS: Low-dose bismuth quadruple therapy proved to be effective with lower costs and amount of antibiotics and bismuth. Gastrus® might be an option for patients with contraindications to bismuth. L. reuteri was unable to significantly counteract dysbiosis induced by antibiotics. How to administer probiotics to prevent gut microbiota alterations remains an open question.

Increased Presence of Antibodies against Type I Interferons and Human Endogenous Retrovirus W in Intensive Care Unit COVID-19 Patients.

In this work, we observed an increased presence of antibodies (Abs) against type I interferon (IFN-I) in coronavirus disease 2019 (COVID-19) patients admitted to the intensive care unit (ICU) compared to non-ICU COVID-19 patients and healthy control (HC) subjects. Human endogenous retrovirus W (HERV-W) can reactivate after viral infection; therefore, we also investigated the presence of antibodies against HERV-W envelope (HERV-W-env)-derived epitopes. A total of 113 subjects (41 female and 72 male subjects) were analyzed. A significant difference in autoantibodies against IFN-α, IFN-ω, and HERV-W was observed between HCs and ICU patients; indeed, the latter have higher levels of autoantibodies against IFN-α, IFN-ω, and HERV-W than subjects with mild COVID-19 and HCs. Neutralizing anti-IFN-I autoantibodies may affect the ability of IFN-I to bind to the type I interferon receptor (IFNAR), blocking the activation of the antiviral response. IMPORTANCE In this work, we report the increased presence of IFN autoantibodies in correlation with HERV-W-env autoantibodies in ICU COVID-19 patients. The novelty of the results is in the association of these IFN autoantibodies with autoantibodies against HERV-W-env, a protein recently discovered to be overexpressed in lymphocytes of COVID-19 patients and correlated with severe disease and pneumonia. Type I IFNs are part of a complex cross-regulatory network; however, in a small percentage of cases, the increase in autoantibodies against these proteins may lead to damage to the host instead of protection against infectious diseases.

Metaproteomic Profile of the Colonic Luminal Microbiota From Patients With Colon Cancer.

Recent studies have provided evidence of interactions among the gut microbiota (GM), local host immune cells, and intestinal tissues in colon carcinogenesis. However, little is known regarding the functions exerted by the GM in colon cancer (CC), particularly with respect to tumor clinical classification and lymphocyte infiltration. In addition, stool, usually employed as a proxy of the GM, cannot fully represent the original complexity of CC microenvironment. Here, we present a pilot study aimed at characterizing the metaproteome of CC-associated colonic luminal contents and identifying its possible associations with CC clinicopathological features. Colonic luminal contents were collected from 24 CC tissue specimens immediately after surgery. Samples were analyzed by shotgun metaproteomics. Almost 30,000 microbial peptides were quantified in the samples, enabling the achievement of the taxonomic and functional profile of the tumor-associated colonic luminal metaproteome. Upon sample aggregation based on tumor stage, grade, or tumor-infiltrating lymphocytes (TILs), peptide sets enabling discrimination of sample groups were identified through discriminant analysis (DA). As a result, Bifidobacterium and Bacteroides fragilis were significantly enriched in high-stage and high-grade CC, respectively. Among metabolic functions, formate-tetrahydrofolate ligase was significantly associated with high-stage CC. Finally, based on the results of this pilot study, we assessed the optimal sample size for differential metaproteomic studies analyzing colonic luminal contents. In conclusion, we provide a detailed picture of the microbial and host components of the colonic luminal proteome and propose promising associations between GM taxonomic/functional features and CC clinicopathological features. Future studies will be needed to verify the prognostic value of these data and to fully exploit the potential of metaproteomics in enhancing our knowledge concerning CC progression.

Systematic identification of NF90 target RNAs by iCLIP analysis.

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. We validated a subset of targets and investigated the impact of NF90 on their expression levels. Two of the top targets, IRF3 and IRF9 mRNAs, encode the proteins IRF3 and IRF9, crucial regulators of the interferon pathway involved in the SARS-CoV-2 immune response. Our results support a role for NF90 in modulating key genes implicated in the immune response and offer insight into the immunological response to the SARS-CoV-2 infection.

Proteomic characterization of Echinococcus granulosus sensu stricto, Taenia hydatigena and Taenia multiceps metacestode cyst fluids.

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.

"Swab Team" in the SARS-CoV-2 outbreak containment among healthcare workers.

INTRODUCTION: To analyze the virus spread among Sassari Hospital staff in the first Covid-19 wave and the impact of the Swab Team, a multidisciplinary task force entitled of nasopharyngeal swab collection and testing. METHODOLOGY: Nasopharyngeal swabs from HCWs between March 6 and May 28 2020 are evaluated. RESULTS: 4919 SARS-CoV-2 tests were performed on 3521 operators. Nurses and doctors are the categories at highest risk. After the Swab Team institution, the average number of swabs raised from 47/day to 86/day (p = 0.007). Positive samples decreased from 18.6% to 1.7% (p < 0.0001). CONCLUSIONS: The Swab Team is effective in increasing the cases tested and in reducing the reporting time. Procedure standardization reduces the risk for all the subjects involved (no transmission among swab team members, nor during the sample collection).

The Metaproteomics Initiative: a coordinated approach for propelling the functional characterization of microbiomes.

Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. The Initiative aims to be the central information hub and open meeting place where newcomers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field. We invite the entire microbiome community to join and discuss potential synergies at the interfaces with other disciplines, and to collectively promote innovative approaches to gain deeper insights into microbiome functions and dynamics. Video Abstract.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Time-restricted feeding induces Lactobacillus- and Akkermansia-specific functional changes in the rat fecal microbiota.

Diet is a key factor influencing gut microbiota (GM) composition and functions, which in turn affect host health. Among dietary regimens, time-restricted (TR) feeding has been associated to numerous health benefits. The impact of TR feeding on the GM composition has been mostly explored by means of metagenomic sequencing. To date, however, little is known about the modulation of GM functions by this dietary regimen. Here, we analyzed the effects of TR feeding on GM functions by evaluating protein expression changes in a rat model through a metaproteomic approach. We observed that TR feeding has a relevant impact on GM functions, specifically leading to an increased abundance of several enzymes involved in carbohydrate and protein metabolism and expressed by Lactobacillus spp. and Akkermansia muciniphila. Taken together, these results contribute to deepening our knowledge about the key relationship between diet, GM, and health.

Effect of Different Disease-Modifying Therapies on Humoral Response to BNT162b2 Vaccine in Sardinian Multiple Sclerosis Patients.

Objectives: Vaccination against COVID-19 is highly recommended to patients affected by multiple sclerosis (MS); however, the impact of MS disease-modifying therapies (DMTs) on the immune response following vaccination has been only partially investigated. Here, we aimed to elucidate the effect of DMTs on the humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines in MS patients. Methods: We obtained sera from 912 Sardinian MS patients and 63 healthy controls 30 days after the second dose of BNT162b2 vaccine and tested them for SARS-CoV-2 response using anti-Spike (S) protein-based serology. Previous SARS-CoV-2 infection was assessed by anti-Nucleocapsid (N) serology. Patients were either untreated or undergoing treatment with a total of 13 different DMTs. Differences between treatment groups comprised of at least 10 patients were assessed by generalized linear mixed-effects model. Demographic and clinical data and smoking status were analyzed as additional factors potentially influencing humoral immunity from COVID-19 vaccine. Results: MS patients treated with natalizumab, teriflunomide, azathioprine, fingolimod, ocrelizumab, and rituximab showed significantly lower humoral responses compared to untreated patients. We did not observe a statistically significant difference in response between patients treated with the other drugs (dimethyl fumarate, interferon, alemtuzumab and glatiramer acetate) and untreated patients. In addition, older age, male sex and active smoking were significantly associated with lower antibody titers against SARS-CoV-2. MS patients previously infected with SARS-CoV-2 had significantly higher humoral responses to vaccine than uninfected patients. Conclusion: Humoral response to BNT162b2 is significantly influenced by the specific DMTs followed by patients, as well as by other factors such as previous SARS-CoV-2 infection, age, sex, and smoking status. These results are important to inform targeted strategies to prevent clinically relevant COVID-19 in MS patients.

Comparative Evaluation of MaxQuant and Proteome Discoverer MS1-Based Protein Quantification Tools.

MS1-based label-free quantification can compare precursor ion peaks across runs, allowing reproducible protein measurements. Among bioinformatic platforms enabling MS1-based quantification, MaxQuant (MQ) is one of the most used, while Proteome Discoverer (PD) has recently introduced the Minora tool. Here, we present a comparative evaluation of six MS1-based quantification methods available in MQ and PD. Intensity (MQ and PD) and area (PD only) of the precursor ion peaks were measured and then subjected or not to normalization. The six methods were applied to data sets simulating various differential proteomics scenarios and covering a wide range of protein abundance ratios and amounts. PD outperformed MQ in terms of quantification yield, dynamic range, and reproducibility, although neither platform reached a fully satisfactory quality of measurements at low-abundance ranges. PD methods including normalization were the most accurate in estimating the abundance ratio between groups and the most sensitive when comparing groups with a narrow abundance ratio; on the contrary, MQ methods generally reached slightly higher specificity, accuracy, and precision values. Moreover, we found that applying an optimized log ratio-based threshold can maximize specificity, accuracy, and precision. Taken together, these results can help researchers choose the most appropriate MS1-based protein quantification strategy for their studies.

A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant.

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.