S. aureus, the genomic basis of clinical phenotypes can be investigated in much more detail, allowing a much deeper understanding of the mechanisms involved in disease. We characterized in detail the S. aureus genomic island vSaβ and defined a superordinate system to categorize S. aureus strains based on their vSaβ type, providing information about the strains’ virulence-associated genes and clinical potential.With the rapid increase of available sequencing data on clinically relevant bacterial species such as  Staphylococcus aureus is a widespread opportunistic pathogen to humans and animals. Of its genome, 20 to 25% varies between strains and consists of phages, pathogenicity islands, transposons, and genomic islands. S. aureus harbors up to three genomic islands, vSaα, vSaβ, and vSaγ. The vSaβ region of S. aureus can encode a number of virulence-associated factors, such as serine proteases, leukocidins, enterotoxins, bacteriocins, or a hyaluronate lyase. In this study, the vSaβ regions of 103 clinically relevant S. aureus strains were characterized in silico and compared to the three predefined vSaβ types. We here suggest a superordinate system of 15 different vSaβ types, of which 12 were newly defined. Each vSaβ type has a distinct structure with a distinct set of genes, which are both highly conserved. Between the different types, gene content and composition vary substantially. Based on our data, a strain’s vSaβ type is strongly coupled with its clonal complex, suggesting that vSaβ was acquired in an ancestral S. aureus strain, arguably by phage mediation, before differentiation into clonal complexes. In addition, we addressed the issue of ambiguous nomenclature in the serine protease gene cluster and propose a novel, phylogeny-based nomenclature of the cluster contained in the vSaβ region.IMPORTANCE With the rapid increase of available sequencing data on clinically relevant bacterial species such as S. aureus, the genomic basis of clinical phenotypes can be investigated in much more detail, allowing a much deeper understanding of the mechanisms involved in disease. We characterized in detail the S. aureus genomic island vSaβ and defined a superordinate system to categorize S. aureus strains based on their vSaβ type, providing information about the strains’ virulence-associated genes and clinical potential. Staphylococcus aureus is a commensal colonizer of the skin and mucous tissue of up to two-thirds of the human population (S. aureus genome consists of the core genome and the accessory (or auxiliary) genome. The core genome, representing about 75%, is conserved in all S. aureus strains and consists largely of genes related to metabolism or other housekeeping functions. The accessory genome, about 20 to 25%, consists of mobile (or formerly mobile) genetic elements (MGE), such as bacteriophages, chromosomal cassettes, pathogenicity islands, transposons, and genomic islands  are available in the NCBI database. Such an enormous increase in sequencing data calls for a new, superordinate system to classify S. aureus strains based on genomic islands. The genomic island vSaβ is of particular interest, as it carries two genes belonging to the type I staphylococcal restriction-modification (RM) system (hsdM and hsdS) and harbors a number of virulence-associated genes, such as a hyaluronate lyase precursor gene (hysA), a lantibiotic gene cluster (bacteriocins of S. aureus [bsa]), two leukocidin genes (lukD and lukE), an enterotoxin gene cluster (EGC), and a cluster of serine protease genes (serine protease like [spl] genes).In 2008, when Baba et al.  with DNA sequence similarities ranging from 42% to 94% , and 66 strains showed novel vSaβ types (IV to XV)  originated mostly from humans, with the exception of 1 strain originating from a bovine mammary infection. vSaβ type I is found commonly and included well-studied clinical strains, such as strains N315, 502A, Mu3, Mu50, and JH1. vSaβ type II (n = 17) comprised 8 human isolates with the well-known strain COL and strain DSM 20231T. The remaining isolates originated from bovine mastitis. Of the vSaβ type III strains (n = 9), 7 originated from humans , and 2 strains were of unknown origin. All vSaβ type IV strains (n = 8) were isolated from bovine mastitis. Of vSaβ type V (n = 9), one host was unknown, and the other 8 strains originated from humans. vSaβ type VI (n = 11) were all isolated from either humans or cattle.vSaβ types VII to XI. vSaβ type VII (n = 5) contained 4 human isolates and 1 bovine mastitis isolate, and for vSaβ type VIII, only a single isolate was found. vSaβ types IX (n = 5) and X (n = 2) were uniquely isolated from ovine mastitis. The two vSaβ type XI strains were bovine mastitis strains. From vSaβ type XII strains (n = 6), 2 originated from cattle and 4 from humans. Out of 9 vSaβ type XIII strains, 5 originated from human hosts and 2 from cattle, and for 2 strains, no information was available. One vSaβ type XIV (n = 6) strain was isolated from Swiss cattle and 5 from human hosts. The two strains with type XV were both bovine mastitis isolates.Only a few strains harbored hp genes). In the strain Mu50 (reference strain for vSaβ type I [see below]) the hp with the locus tag SAV1803 marks the 5′ terminus of vSaβ, and SAV1831 marks the 3′ terminus. These two hp genes could be found in nearly all vSaβ islands .All vSaβ islands were enclosed by 2 distinct genes coding for hypothetical proteins  carrying a distinct set of virulence genes  genes, the RM genes hsdM and hsdS, a cluster of spl genes , a probable beta-lactamase gene (bla), a cluster of 9 bsa genes , the lukD and lukE genes, as well as an EGC consisting of the seg, sei, sem, sen, seo, and seu genes. In some cases, the seu gene was replaced by the two truncated genes, ϕent1 and ϕent2. The compositions of these genes and gene clusters varied substantially between different vSaβ types but were highly conserved within the same vSaβ type  pipeline.Most ce genes . They inSaβ type . In addivSaβ islands and aligning all related sequences, a consensus structure for each vSaβ type could be defined. vSaβ type I possessed the genes of the type I RM system, an spl cluster , lukD, lukE, and a complete EGC. The amino acid sequences were, with only very few exceptions, highly similar within this vSaβ type (>95%) , a bsaG fragment, bla, and remnants of phages were detected , a bla, a bsa cluster, and the lukD and lukE genes. Exceptions were some strains that harbored truncated hsdM, basE, and lukE genes. Moreover, vSaβ type II comprised sequences for two mep genes, bla, a number of hp genes, and a partial phage , and an EGC. Besides three spl genes being all pseudogenes , vSaβ type III lacked lukD and lukE but had additional genes in its 5′ region . vSaβ type III carried sequences for mep, hp, and remnants of a phage. In most of the type III strains, the core proteins were highly conserved, showing an identity of ≥99% when S. aureus strain MRSA252 was used as a reference. Deviations thereof could be found in the spl cluster and in hsdM , a complete bsa cluster, an EGC (with truncated seg and sem), lukD, lukE, and the bla gene. hsdM and hsdS were either missing or had premature stop codons. The amino acid sequences of all other vSaβ key genes were highly conserved (≥99%) among strains, with only a small number of exceptions (NC011614) was detected in 6 vSaβ type IV strains, coding for approximately 75 proteins including those encoded by the EGC also found in other vSaβ types. In contrast, the strains M2323C and Sa110 harbored only remnants of two phages; hence, the vSaβ islands of these two strains were shorter than the other type IV islands. The structure of vSaβ type V differed substantially from the other types and was only 15,965 bp long and highly conserved, showing a nucleotide sequence identity of ≥99% when using strain S0385 as a reference. From the typical genes, vSaβ type V harbored only hysA. However, it carries a total of 4 transposons, as well as a number of hp genes, some of which were related to FIG numbers, or they were identified as lpn genes. Within vSaβ type VI, strain K2R was a suitable representative. It comprised hysA, hsdS, hsdM, an spl cluster , bla, a bsaG fragment, lukD, lukE, a phage integrase, as well as a number of hp genes, some of which were assigned a FIG number, and others were identified as lpn genes. In addition, a partial phage was detected encompassing the entire spl cluster. Four strains  had a frameshift in the beginning of lukE, leading to a truncated protein. Additionally, G07I and Lodi4R also lacked the 1splD and the second 2splD gene. Except for these very few special cases, all genes of vSaβ type VI were highly conserved between strains, showing an amino acid identity of ≥98%. The low overall nucleotide identity of 76% in strain Lodi4R compared to the reference was based on the insertion of the transposon Tn554 within hysA. This strain was, therefore, considered a special case of vSaβ type VI. Tn554 carried 3 transposase genes, as well as a bla operon carrying the blaI, blaR1, and blaZ. If Tn554 was disregarded, the nucleotide sequence identity increased to 93%. vSaβ type VII, similar to type V, was short  and lacked the typical vSaβ genes, except for hysA and a hsdS fragment. Furthermore, all vSaβ type VII strains showed a partial phage with genes for proteins targeting the host’s immune response (chemotaxis-inhibiting protein Cpn and extracellular complement-binding protein Scn), as well as genes for a peptidase, a phage lysine, an ATP-binding protein, and a transposase. The amino acid sequences of these phage-encoded proteins were identical, with the exception of M3386D that was lacking a part of the phage containing genes coding for the ATP-binding protein and the transposase. The vSaβ type VIII included only the JKD6159 strain. It comprised hysA, hsdM, hsdS, an spl cluster , bla, a bsaG fragment, lukD, lukE, a partial phage, a number of hp and phage-related genes, and a number of lpn genes in the 5′ region. For vSaβ type IX, ED133 served as the reference harboring the vSaβ key genes hsdM and hsdS, an spl cluster , lukD, lukE (truncated), bla, and a complete bsa cluster, as well as a number of hp genes, some of which were identified as lpn genes and others that were assigned a FIG number. In addition, vSaβ type IX carried 2 genes encoding ATP-binding cassette (ABC) transporter proteins. and a gene encoding a phage integrase. Despite the isolates being from 3 different countries, they shared an overall nucleotide sequence identity of ≥95% in their vSaβ. The only small difference in the vSaβ key genes was found in hsdM and lukD, which showed lower similarities in some strains , a bla, a bsaG fragment, lukD, lukE, a complete hsdS gene and an hsdM gene with a premature stop codon. In addition to a number of hp and FIG-assigned genes, lpn, two genes encoding an ABC transporter, and a phage integrase gene could be found, similar to vSaβ type IX. The amino acid identity in the key genes were ≥98%, with the exceptions of lukE being truncated in O11, 1splD lacking in strain O46, and a slightly lower identity in 2splD (93% amino acid identity). vSaβ type XI consisted of two strains with an amino acid identity of ≥99% in the vSaβ key genes and a nucleotide identity of 99% over the entire vSaβ region. Their vSaβ consists of hsdM and hsdS, but hsdM has a premature stop codon. Furthermore, vSaβ type XI was characterized by an spl cluster , a bla, a basG fragment, lukD, lukE, a complete EGC (with a truncated sen), several hp genes (some of which with assigned FIG numbers), and a short hysA fragment. The most striking feature of vSaβ type XII was the enlarged spl cluster consisting of splA, splB, splC, 1splD, 2splD, 3splD, and 2 copies of 4splD. It further harbored both RM genes (hsdM with premature stop codon), bla, a bsaG fragment, lukD, lukE, and a complete EGC along with several hp genes, some with assigned FIG numbers. The strains categorized as vSaβ type XIII had a typical vSaβ structure in the 3′ end, yet a clear beginning could not be defined for this vSaβ type, as it lacked the conserved 5′-end sav1803. Instead, there were three hp genes as well as two recently discovered enterotoxin sequences, sel26 and sel27. These were followed by several hp genes (some with assigned FIG number) and genes typical for vSaβ, such as lpn, hsdM, hdsS, bla, the EGC followed by the tRNA cluster, and sav1831, typically marking the 3′ end. vSaβ type XIV harbored hysA, both RM genes (hsdM and hsdS), an spl cluster , bla, a bsaG fragment, lukD, and lukE. When strain G08M was used as a reference, most proteins showed an amino acid identity of 100%, with the exceptions being three strains with truncated hsdM genes. vSaβ type XV consisted of both RM genes (hsdM and hsdS), an spl cluster , bla, a bsaG fragment, lukD, and lukE. When strain Lodi11bM was used as a reference, the only differences were lukE being truncated and SplD4 having a lower amino acid identity in strain 4185.By typing the e (>95%) . The exc strains . Furtherdetected ; Fig. 1.al phage . The con in hsdM . The vSaceptions . Further strains . vSaβ tyvSaβ types had the same basic structural design. The design was minimal for types V, VII, and XIII and was more complex for the other types, as they harbored a distinct set of virulence factors and gene clusters. Also common to all vSaβ types, except to type XIII, was the pervasive flanking by the same conserved sequences with the locus tags SAV1803 and SAV1831. Variation among the vSaβ types was observed for the spl cluster in gene number and composition. Furthermore, some types were characterized by truncated RM genes and/or bsa or spl genes. Within each vSaβ type, they were highly conserved, at the structural and the protein levels.In summary, all vSaβ. Hence, categorization of vSaβ based on the nucleotide sequence identity of the entire genomic island can be considered the method of choice when the entire sequence of the genomic island is available.Inspection of the HsdS sequences with a phylogenetic approach  revealedvSaβ islands, with the exceptions of types V, VII, and XIII, carried an spl cluster, harboring between 4 and 9 spl genes. Phylogenetic analyses using the maximum-likelihood method confirmed the existing Spl family members SplA, SplB, SplC, and SplD, but with SplD having 4 different variants   . SplD1 rspa typing showed that one vSaβ type can harbor a number of spa types, but each spa type is limited to one vSaβ type. Highly striking was the consistency of a vSaβ type with the strains’ clonality. The predominance of a single clonal complex (CC) per vSaβ type underlined the concept that vSaβ acquisition and its diversification happened prior or simultaneously to the clonal diversification of ancestral S. aureus strains.S. aureus sequences deposited in databases is growing exponentially. These databases offer a powerful information source for studying the variabilities and consistencies between different isolates and their potential ability to cause disease. As opposed to the slow accumulation of point mutations, acquisition of larger parts of DNA through horizontal gene transfer leads to a rapid genetic change. This can be crucial in survival under certain selection pressures (antibiotics) or in novel niches (new host)  , 12, 13.vSaβ types, the strains belonging to one vSaβ type almost exclusively harbor the same virulence genes. In addition to the gene composition, an amino acid sequence identity of 95% or higher is another characteristic within each type, and in many cases, even 100% over all strains of a vSaβ type , the bsa cluster only (II and IX), or lack both. In this sense, vSaβ type IV can be considered the most complete of all vSaβ types regarding virulence-related gene content and phage. Moon et al. (vSaβ type IV) can be mobilized in vitro, resulting in heterogeneous, yet overlapping particles that integrated sequentially through recombination into the host; in some cases, it even resulted in the transfer of the almost-complete vSaβ , likely just prior or simultaneously to clone formation, resulting in the types observed today.In total, eviously , but by vSaβ types and CCs are in perfect agreement with the topology of the phylogenetic tree by Boss et al. , lpn, and hysA. Interestingly, type VII encodes different virulence factors that tackle the host’s immune defenses. As vSaβ types V and VII have additional transposase genes, we suggest that in these two types, the vSaβ key genes were replaced by a transposon, indicating that vSaβ themselves may be a hot spot for inserting mobile genetic elements and potentially accumulating virulence factors.The discrepancy between the overall structures of vSaβ type XIII has a deviate structure, as it lacks the typical 5′ region found in the other types. Together with types V and VII, it lacks the spl cluster and the luk genes. We cannot exactly locate the beginning of this vSaβ type on the genome, but interestingly, we found two very recently discovered phage-associated enterotoxin genes (sel26 and sel27) in that region (hp (FIG01108398) which is adjacent to the SAV1803 in some other types.t region . FurtherS. aureus strain on a given host and its disease-causing potential, as these regions encode a number of virulence factors . Both proteins are members of the leukocidin family  and 4 gene variants of splD . SplA, SplB, and SplC form their own clades and are clearly separated from the SplD clade  are antimicrobial peptides produced by some Gram-positive bacteria against closely related species , 35 and vSaβ types, it has been suggested that the vSaβ type is dependent on the strain’s HsdS sequence  were excluded from the data set. Hence, our results are limited to these invasive strains only, yet we did find that a few colonizers also fit into the system proposed here (data not shown). In the future, more vSaβ regions need to be analyzed spanning more hosts and including noninvasive strains.In the scope of this study, we limited the data set to clinically relevant vSaβ islands harbor a number of virulence-associated and pathogenic genes with different scopes of action. While the exact functions of many of these genes are yet to be unraveled, it is clear that they have severe effects on the host’s health and are likely to play key roles in S. aureus adaptation to the clinical microenvironment. Our data support a viral origin of the vSaβ region. As the vSaβ type is strongly linked to a strain’s CC, acquisition of the vSaβ region happened in a very ancestral S. aureus strain while the transformation to the distinct vSaβ subtypes occurred before or simultaneously to diversification into the different clones. The here-suggested superordinate system to classify S. aureus strains based on their vSaβ region may be used in the future to assess the clinical potential of an S. aureus strain. In the future, more vSaβ genomic islands need to be analyzed and categorized into this superordinate system.In general, the S. aureus genome collection includes 23 strains that were previously sampled from bovine mastitis  technology and SPAdes v.3.1  and P. Cremonesi .The in-house mastitis . All stres v.3.1  for the vSaβ islands of our 27 bovine mastitis S. aureus genomes as described by Baba et al.  database (https://www.ncbi.nlm.nih.gov/nucleotide/) using the default BLAST settings and limiting the search to S. aureus. To increase the diversity of the vSaβ islands, the NCBI databases were also evaluated for genomes or contigs of clinical S. aureus strains from hosts other than humans or cattle. This approach was selected, as invasive strains of S. aureus are host specific . All available chromosomal sequences were then retrieved, including all contigs containing a complete vSaβ region.In an attempt to characterize the a et al. , it turnT search  of each specific , 38, 39,Using this approach, a total of 76 sequences were obtained, with 43 sequences from genomes and 33 sequences from contigs (see File S2).(i) Data set. In total, 27 of our own and 76 publicly available sequences were included in the present study. From these 103 analyzed S. aureus strains, 58 originated from human hosts, 33 were from bovine hosts, and 7 were from ovine hosts. For 5 strains, no host information was available. Of these 103 strains, 37 strains showed known vSaβ types (I to III), and 66 strains showed novel vSaβ types (IV to XV) (see File S2 for details).vSaβ typing.(ii)  The vSaβ regions were identified on genome sequences by aligning the sequences of conserved hp genes flanking vSaβ at the 5′ end (SAV1803) and 3′ end (SAV1831)  HsdS phylogenetics. To infer the phylogeny of the HsdS proteins (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) for visual inspection and manual curation. Afterwards, the curated MSA was imported into the MEGA X software  Multilocus and  Multilocus sequence typing (MLST) of seven housekeeping genes (spa typing (S. aureus genomes using the Center for Genomic Epidemiology online platform (http://www.genomicepidemiology.org/). In the eBURST V3 program, the sequence types (STs) resulting from the MLST were used to allocate each strain to a clonal complex (CC).ng genes  and spa a typing  were per(v) Annotations. Annotations were assigned to the vSaβ regions using the RAST pipeline (–hp genes with or without a FIG number assigned by the RAST pipeline) were verified by a BLAST search against the UniProt database  Phage detection. Phages and phage remnants were identified by the online software Phage Search Tool Enhanced Release (PHASTER)  , 50. To (vii) Serine protease nomenclature. All complete and curated spl sequences were analyzed and translated into proteins using the standard code. Sixty-eight amino acid sequences were unique  and were used to reconstruct the phylogeny using the same methods as mentioned above for the HsdS protein sequence   of ≥95 and a phylogenetic distance (d) of >0.10. All leaves forming this clade obtain the same letter  added to the basic name of the protein (in this case “Spl”), resulting in the name SplD for all of these leaves. The branch is then followed toward the leaves. If a node is reached forming a monophyletic subclade whose branch connecting to the predecessor is characterized by a BT of ≥95 and d of >0.10, the leaves of this subclade are labeled by adding a number , resulting in the name SplD2. At this stage, further evaluation toward the leaves is stopped. If only one branch of a bifurcation fulfills these conditions, the subclade formed by the nonfulfilling branch gets a new letter or number as well.g sites)  and B. APRJNA531079 with the accession numbers SZYI00000000 (Lodi13K), SZYM00000000 (G12B), SZYO00000000 (G29N), SZYT00000000 (Lodi10B), VCQP00000000 (M2084B), SZYY00000000 (M2130B), SZZA00000000 (M2529B), SZZF00000000 (M5512B), VCQQ00000000 (M5171B), SZZG00000000 (M6020B), SZZE00000000 (M3783C), SZZC00000000 (M2839C), SZYW00000000 (M1280C), SZYX00000000 (M1655C), SZYZ00000000 (M2323C), SZZB00000000 (M2682A), SZYS00000000 (K2R), SZYJ00000000 (G07I), SZYV00000000 (Lodi4R), SZZD00000000 (M3386D), SZYQ00000000 (G42G), SZYP00000000 (G33O), SZYR00000000 (G68P), SZYL00000000 (G11F), SZYN00000000 (G19F), SZYK00000000 (G08M), and SZYU00000000 (Lodi11bM).All whole-genome projects have been deposited in the NCBI database under the BioProject number


Studies evaluating the impacts of parity on LFD of healthy females presented controversial conclusions.To compare the LFD of healthy females broken down according to their parities.1 (n = 34): ≤ 6; G2 (n = 32): > 6] and three classes  of parities were identified. LFD ] were determined. Student’s t-test and ANOVA test with post-Hoc test were used to compare the two groups’ and the three classes’ data.A medical questionnaire was administered and anthropometric data were determined. Two groups [G1 and G2 were age and height matched; however, compared to G1, G2 had a lower body mass index (BMI). C1, C2 and C3 were height, weight and BMI matched; however, compared to C2, C3 was older. G1 and G2 had similar values of FEV1, forced- and slow- vital capacities , maximal mid-expiratory flow (MMEF), forced expiratory flow at x% of FVC (FEFx%), peak expiratory flow (PEF), expiratory and inspiratory reserve volumes , inspiratory capacity (IC), sRaw, FEV1/FVC, FEV1/SVC, and residual volume/total lung capacity (RV/TLC). The three classes had similar values of MMEF, FEFx%, PEF, thoracic gas volume (TGV), ERV, IRV, FEV1/FVC, FEV1/SVC and RV/TLC. Compared to G1, G2 had higher TGV (2.68±0.43 vs. 3.00±0.47 L), RV (1.80±0.29 vs. 2.04±0.33 L) and TLC (4.77±0.62 vs. 5.11±0.67 L). Compared to C1, C2 had higher FEV1 (2.14±0.56 vs. 2.47±0.33 L), FVC (2.72±0.65 vs. 3.19±0.41 L), SVC (2.74±0.61 vs. 3.24±0.41 L), TLC (4.47±0.59 vs. 5.10±0.58 L), IC (1.92±0.41 vs. 2.34±0.39 L) and sRaw (4.70±1.32 vs. 5.75±1.18 kPa*s). Compared to C1, C3 had higher TLC (4.47±0.59 vs. 5.05±0.68 L) and RV (1.75±0.29 vs. 2.04±0.30 L).GIncreasing parity induced a tendency towards lung-hyperinflation. Respiratory aging, which would start from the age of 35–40 , can be eg, age, height, weight and body mass index (BMI)]    , 7, 8. Ox (BMI)] . Accordi status] , 10. Amo status] –22. On t status] . For exa status]  , Tunisid Mali]    with co and FVC ] or of N and FVC ] In the low rate , 13], orow (PEF) ]. Moreov1: parity ≤ 6; G2: parity > 6] and three classes . The second aim was to determine the relationship between parity and some LFD.In view of the above divergence between studies, the main aim of the present study was to compare the LFD of healthy North-African females broken down according to their parities into two groups  or previous hospitalization for pulmonary or thoracic problems; (ii) cardiac diseases that may affect the respiratory system ; (iii) current-smoker or ex-smoker of more than one pack-year; (iv) leanness and obesity stage 2 and more; and (v) high physical-activity level .The total population was divided into four groups: a group for the development of plethysmographic norms specific to northern Algeria’ population , a group for the validation of the GLI-2012 spirometric norms  , a group1 (parity ≤ 6); G2 (parity > 6)] and three classes   , 29–31. ity ≥ 9] . Females2) was calculated. Obesity status was categorized into leanness (BMI < 18.5), normal weight (18.5 ≤ BMI ≤ 24.9), overweight (25.0 ≤ BMI ≤ 29.9), and obesity stages 1 (30.0 ≤ BMI ≤ 34.9), 2 (35.0 ≤ BMI ≤ 39.9) and 3 (BMI > 40.0) , volumes and capacities , ratios , and specific airway resistance .LFD were determined by one qualified person  evaluated the associations between parity, and plethysmographic and anthropometric data. “r2” was considered as “clinically significant” when it was > 0.30 . Among the 244 females included in the whole project, 121 were excluded from this study (97 were under 40 years of age and 24 were nulliparous). The parity’ mean±SD of the remaining 123 females aged ≥ 40 with at least one parity was 6.4±3.5. Based on this data, two groups were randomly chosen to be age- and height- matched . This sdiseases , 47, leadiseases . Lung-hydiseases , 49.1 and FVC  favorably affects the LFD of Nigerians, with increases in both FEV1 and FVC http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Overall this is a well written paper using sound methodologies. There are a few issues, however, in its current form.Major comments:While the authors spend a significant amount of time attempting to explain why they chose parity less than or equal to 6 as their definition of low parity, it still seems like it includes a high number. The authors mention in their introduction that parity is high in Africa but give reference to numbers which would be otherwise considered in the “low parity group,”( i.e everyone less than 6). Also in the results section they mention previous studies and different parity cutoffs which are all lower than the current study.- Why not divide into 3 groups? 1-4, 5-8, 9-12 for instance.- Can there be more information on the health of the females. You mention education, access to food and education, and that the average was 0.75. Then you mention in the discussion that SL, SEL, PAL cannot be identified. Why was this? Is this information included in the study?- Higher parity can be associated with lower economic status. How do you adjust for that in your study?Minor comments:- “Since the different aspects of LFD’ decline are still inescapable [5], and since, the determination of numerous factors of LFD’ decline is not yet closed” – I don’t understand what closed means- “while the total fertility rate in Algeria declined from 1951 to 2015 (from 7.279 to 2.839 or 3.100 children [42]), it remained still higher in some author African countries” -> I think you mean “other”Reviewer #2: RE: PONE-D-19-16514 -- Parity and ageing of the respiratory systemStudies evaluating the impacts of parity on lung function data (LFD) of healthy females present controversial conclusions. Respiratory aging, can be estimated by lung function data (LFD), recognized as predictors of mortality and morbidity. These relationships are based on the subjects’ ethnicity and are included as influencing factors .Parity has been proposed as an additional influence in some studies. Contrary to European and North American studies, parity is of particular impact in low- and lower middle-income countries such as in Africa. In several African countries, recent values of parity were higher than six. A few studies, published between 1999 and 2018, raised the impacts of parity on healthy females’ LFD  with conflicting results. Some studies concluded that high parity was positively associated LFD while others concluded that it was associated with negative effects on LFD.Thus, the purpose of this investigation was to compare lung function data (LFD) of two groups of healthy females divided according to their parities, and to determine the relationship between parity and LFD.Methods. A medical questionnaire was administered and anthropometric data were determined. Parity was introduced as numeric and as dichotomous . LFD ] was performed. Correlation-coefficient (r) and Student’s t-test were used, respectively, to evaluate therelationships between parity and LFD  and to compare the two groups’ mean±SD quantitative data.Main results: Compared to GI, G2 had a lower mean body mass index (30.vs. 27.6) and a higher parity (4 vs. 10). G1 and G2 had similar values of forced expiratory volume in 1 s (FEV1), forced- and low- vital capacity , maximal mid-expiratory flow, forced expiratory flow at x% of FVC, peak expiratory flow, expiratory and inspiratory reserve volumes, inspiratory capacity, sRaw, FEV1/FVC, FEV1/SVC, and residual volume/total lung capacity (RV/TLC). Compared to G1, G2 had significantly higher thoracic gas volume, RV and TLC. In the total sample, significant positive correlations were found between parity and TGV (r=0.31), RV (r=0.33) and TLC (r=0.25).The authors concluded that increasing parity induces a tendency towards lung-hyperinflation.General comments:A key goal of this study should be to describe and explain the differences between high and low parity women, and explain the differences found between the two cohorts and the mechanisms that would explain the findings. Instead, the authors launch into a lengthy disquisition of why lung function testing is obtained in general, how lung function changes with age and other factors , how cohort values are compared to controls, and a detailed description of differences in lung function amongst European, American and northern (the Sahel) and sub-Saharan African ethnic groups. This is interesting material but has little to do with the main purpose of the study which was ostensibly to assess the effects of parity on lung function (and what is implied in the title of the paper). The authors should have primarily focused on this topic. The rest of their discussion is better suited for a textbook or monograph on the epidemiology of lung function.Not only that, but the paper is needlessly prolonged by repeating in the discussion much of what’s already stated in the introduction. The explanation for the changes in LFD is only briefly explained in a paragraph ending on p. 23. Instead of placing most of this information in the supplementary material it should be included as part of the main discussion – that is the main point of the paper.Specific comments:The paper needs grammatical and stylistic corrections. I will list a few:1. Pages and lines should be numbered – makes changes easily traceable.2. The authors should reconsider the weightedness and clinical importance of their correlation coefficients. From a clinically relevant standpoint, the correlation coefficient should be squared: an r-squared value of >0.3 is considered to be clinically relevant. Thus r=0.3 becomes 0.09, which is not clinically meaningful; r=0.4 becomes 0.16, also not relevant. In other words, what may be statistically significant may not be clinically significant or relevant.3. Intro., l. 8, “…next line: “…ongoing...” instead of “… not yet closed…”4. Next line: Instead of “…guaranteed...”, state “…has been validated…”5. Intro, next page, l. 5: Chad is repeated in same sentence.6. Same page, l. 7: For Tunisia there are 4 references cited, not 3.7. Intro, last paragraph, l. 1: “In view of…” instead of “…in front of…”8. Under plethysmographic measurements, l. 1; should read “Lung volume data were determined by… plethysmography (Body-box 5500…)".9. Under discussion, last para, last line: “… still higher in other African countries.”10. Under Methodology discussion, l. 8: “…who reported being healthy…”11. Same section, next 2 pages: Beginning with “The non-inclusion criteria were respected.” all the way through the next page, ending with “… can’t be explained by their menopause status” should be deleted, as it is repetitious from the introduction and also not relevant to the discussion itself.12. Results discussion, last 2 pages: Beginning with “The present study correlations…”, the authors just regurgitate what is already listed in the supplementary tables – this information should be transferred to the results section and not repeated here. Rather, the authors should expand on and explain their findings from a physiological standpoint, that is, how are the findings explained. This has much to do with physiologic differences of the thoracic cage between men and women and how pregnancy affects these mechanical properties during pregnancy and with repeated pregnancies. This would add a unique aspect to their discussion because there is not much information on the effects of multiparity on respiratory mechanics.13. Again, these r-values should be squared to determine if they are truly clinically significant – many of the LFD changes will turn out to be not significant or relevant to parity.14. Last page, last para.: This brief paragraph should be greatly expanded to explain the effects of multiparity on respiratory function , particularly in regards to the lung hyperinflation – how is this event linked to changes in the thoracic cage? In fact, this expanded discussion should nearly completely replace the bloated epidemiologic data from different countries listed by the authors. Pregnancy likely affects the fundamental changes that occur in the respiratory system in a common way, with only subtle differences amongst ethnic/national groups.In short, the information provided here is interesting, and relatively new and should be reported, but in a greatly revised form. It can be presented in a cleaner, more concise manner with greater emphasis placed on the physiologic explanations rather than just reporting epidemiologic data from different countries. The latter aspect can be considerably shortened. Finally, the clinical relevance of statistically significant correlations should be re-considered in a clinically relevant manner.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, <gdiv></gdiv> 25 Sep 2019TO THE ACADEMIC EDITORDear EDITOR,Thank you for giving us the chance to review and improve our paper. Please find below the responses to the two reviewers questions/suggestions. Sincerely yours.TO REVIEWER #1Dear Reviewer,Thank you for your comments. Please find below the responses to your questions/suggestions. Sincerely,REMARK N°1Overall this is a well written paper using sound methodologies. There are a few issues, however, in its current form. CORRECTIVE ACTIONAll your suggestions were applied.REMARK N°2 a*While the authors spend a significant amount of time attempting to explain why they chose parity less than or equal to 6 as their definition of low parity, it still seems like it includes a high number. The authors mention in their introduction that parity is high in Africa but give reference to numbers which would be otherwise considered in the “low parity group,”( i.e everyone less than 6). **Also in the results section they mention previous studies and different parity cutoffs which are all lower than the current study. Why not divide into 3 groups? 1-4, 5-8, 9-12 for instance.RESPONSE*Needed changes were applied over all the paper.**We have takin into account your suggestion to divide the population into three 3 classes: Parity 1-4: n=15Parity 5-8: n=28Parity 9-14: n=23CORRECTIVE ACTION*The following sentence was deleted from the “Introduction” “”.**In the “Methods” section, we have added that our population was divided into two groups (Table 1) and into three classes (Table 2) of parity.*We have also applied all needed changes in the text .REMARK N°2 bCan there be more information on the health of the females. You mention education, access to food and education, and that the average was 0.75. Then you mention in the discussion that SL, SEL, PAL cannot be identified. Why was this? Is this information included in the study?RESPONSEIn the manuscript (L185-187), we have noted that “The population was relatively homogeneous and considered as belonging to the middle class with an elevated human development index of 0.75 [27].” The information was retrieved from the following reference (ref 27): Human development index in Algeria. Therefore, SL, SEL, PAL weren’t objectively determined.CORRECTIVE ACTIONEducation (schooling level) was addressed as a limitation study (L303-306): “First, compared to females with a high schooling-level, those with a low level had higher parity [20] and lower LFD [6]. However, LFD differences concerned only peripheral flows such as PEF (increase by 254 mL/s) and FEF25% (increase by 150 mL/s) [6].”REMARK N°2 cHigher parity can be associated with lower economic status. How do you adjust for that in your study?RESPONSEWe agree with your remark. The association between high parity and lower socioeconomic level was highlighted as a study limitation (L306-311).CORRECTIVE ACTIONThe following sentence is noted in the paper (L306-311): “Secondly, in high-income countries, compared to females with a high socioeconomic-level, those with a low one had higher parity [20], lower LFD , and a tendency towards a distal OVD [44]. Moreover, in Polish females with COPD, the FEV1 decline’ acceleration with increased parity (≥ 4) was accounted for by the low socioeconomic-level of the latter [20]. However, it seemed that North-African females’ LFD weren’t influenced by the socioeconomic-level [6].”REMARK N°3 aSince the different aspects of LFD’ decline are still inescapable [5], and since, the determination of numerous factors of LFD’ decline is not yet closed” – I don’t understand what closed meansRESPONSEThe sentence was corrected (as suggested by the 2nd reviewer: please see his remark 9c)CORRECTIVE ACTION“ongoing” replaced “not yet closed” L110.REMARK N°3 bwhile the total fertility rate in Algeria declined from 1951 to 2015 (from 7.279 to 2.839 or 3.100 children [42]), it remained still higher in some author African countries” -> I think you mean “other”RESPONSECorrection done.CORRECTIVE ACTIONPlease see L292TO REVIEWER #2Dear Reviewer,Thank you for your comments. Please find below the responses to your questions/suggestions. Sincerely yours.REMARK N°1Studies evaluating the impacts of parity on lung function data (LFD) of healthy females present controversial conclusions. Respiratory aging, can be estimated by lung function data (LFD), recognized as predictors of mortality and morbidity. These relationships are based on the subjects’ ethnicity and are included as influencing factors .Parity has been proposed as an additional influence in some studies. Contrary to European and North American studies, parity is of particular impact in low- and lower middle-income countries such as in Africa. In several African countries, recent values of parity were higher than six. A few studies, published between 1999 and 2018, raised the impacts of parity on healthy females’ LFD  with conflicting results. Some studies concluded that high parity was positively associated LFD while others concluded that it was associated with negative effects on LFD.Thus, the purpose of this investigation was to compare lung function data (LFD) of two groups of healthy females divided according to their parities, and to determine the relationship between parity and LFD.Methods. A medical questionnaire was administered and anthropometric data were determined. Parity was introduced as numeric and as dichotomous . LFD ] was performed. Correlation-coefficient (r) and Student’s t-test were used, respectively, to evaluate the relationships between parity and LFD  and to compare the two groups’ mean±SD quantitative data.Main results: Compared to GI, G2 had a lower mean body mass index (30.vs. 27.6) and a higher parity (4 vs. 10). G1 and G2 had similar values of forced expiratory volume in 1 s (FEV1), forced- and low- vital capacity , maximal mid-expiratory flow, forced expiratory flow at x% of FVC, peak expiratory flow, expiratory and inspiratory reserve volumes, inspiratory capacity, sRaw, FEV1/FVC, FEV1/SVC, and residual volume/total lung capacity (RV/TLC). Compared to G1, G2 had significantly higher thoracic gas volume, RV and TLC. In the total sample, significant positive correlations were found between parity and TGV (r=0.31), RV (r=0.33) and TLC (r=0.25).The authors concluded that increasing parity induces a tendency towards lung-hyperinflation.RESPONSENo corrective action is needed in this stage. Please note that we have applied all your asked corrections.REMARK N°2aA key goal of this study should be to describe and explain the differences between high and low parity women, and explain the differences found between the two cohorts and the mechanisms that would explain the findings. Instead, the authors launch into a lengthy disquisition of why lung function testing is obtained in general, how lung function changes with age and other factors , how cohort values are compared to controls, and a detailed description of differences in lung function amongst European, American and northern (the Sahel) and sub-Saharan African ethnic groups. This is interesting material but has little to do with the main purpose of the study which was ostensibly to assess the effects of parity on lung function (and what is implied in the title of the paper). The authors should have primarily focused on this topic. The rest of their discussion is better suited for a textbook or monograph on the epidemiology of lung function.RESPONSESeveral changes were performed in the Discussion section. CORRECTIVE ACTION*The paper is now primarily focused on the effects of parity on lung function data. **Several parts from the “Methodology discussion” subsection was moved to the S3 File.REMARK N°2bNot only that, but the paper is needlessly prolonged by repeating in the discussion much of what’s already stated in the introduction. The explanation for the changes in LFD is only briefly explained in a paragraph ending on p. 23. Instead of placing most of this information in the supplementary material it should be included as part of the main discussion – that is the main point of the paper.RESPONSESeveral changes were performed in the Discussion.CORRECTIVE ACTIONThe answer to the following question “How to explain the impacts of parity on LFD?” is moved from the S3 File to the main manuscript (L381-428).REMARK N°3aPages and lines should be numbered – makes changes easily traceable.CORRECTIVE ACTIONPages and lines are added.REMARK N°3bThe authors should reconsider the weightiness and clinical importance of their correlation coefficients. From a clinically relevant standpoint, the correlation coefficient should be squared: an r-squared value of >0.3 is considered to be clinically relevant. Thus r=0.3 becomes 0.09, which is not clinically meaningful; r=0.4 becomes 0.16, also not relevant. In other words, what may be statistically significant may not be clinically significant or relevant.RESPONSEAll your suggestions were applied.CORRECTIVE ACTION*In the ‘statistical analysis” subsection, the following sentence was added (L239-241): “Determination-coefficient (r2 = square of the Pearson product-moment correlation-coefficient) evaluated the associations between parity, and plethysmographic and anthropometric data. “r2” was considered as “clinically significant” when it was > 0.30 [37]” **Table 3 exposes the “Determination coefficient (r2) between anthropometric and plethysmographic data, and parity.” Only “clinically significant” correlations were considered.***All required changes were applied in the main manuscript.REMARK N°3cIntro., l. 8, “…next line: “…ongoing...” instead of “… not yet closed…”RESPONSEAgree with youCORRECTIVE ACTIONDone L110.REMARK N°3dNext line: Instead of “…guaranteed...”, state “…has been validated…”RESPONSEAgree with youCORRECTIVE ACTIONDone L112.REMARK N°3eIntro, next page, l. 5: Chad is repeated in same sentence.RESPONSEAgree with youCORRECTIVE ACTIONThe second ‘Chad” was deleted (L129).REMARK N°3fSame page, l. 7: For Tunisia there are 4 references cited, not 3.RESPONSEReferences 12 :491-503) and 13 :789-804) are derived from the same project and described the same population.CORRECTIVE ACTIONOnly reference 12 was kept.REMARK N°3gIntro, last paragraph, l. 1: “In view of…” instead of “…in front of…”RESPONSEAgree with your suggestion.CORRECTIVE ACTIONDone L143.REMARK N°3hUnder plethysmographic measurements, l. 1; should read “Lung volume data were determined by… plethysmography (Body-box 5500…)".RESPONSEAgree with your suggestion.CORRECTIVE ACTIONThe following sentence was added (L218-219): “LFD were determined by one qualified person (AK in the authors’ list) via a plethysmograph . The latter was calibrated each morning.”REMARK N°3iUnder discussion, last para, last line: “… still higher in other African countries.”RESPONSEAgree with your suggestion.CORRECTIVE ACTIONDone L292.REMARK N°3kUnder Methodology discussion, l. 8: “…who reported being healthy…”RESPONSEAgree with your suggestion.CORRECTIVE ACTIONNote: the “Methodology discussion“ subsection was moved to the S3 File.REMARK N°3lSame section, next 2 pages: Beginning with “The non-inclusion criteria were respected.” all the way through the next page, ending with “… can’t be explained by their menopause status” should be deleted, as it is repetitious from the introduction and also not relevant to the discussion itself.RESPONSEWe have considered your suggestion and we agree with it.CORRECTIVE ACTIONIn order to shorten the manuscript, all the long sentence “The non-inclusion criteria were respected……… can’t be explained by their menopause status” was moved to the S3 File.REMARK N°3m*Results discussion, last 2 pages: Beginning with “The present study correlations…”, the authors just regurgitate what is already listed in the supplementary tables – this information should be transferred to the results section and not repeated here. **Rather, the authors should expand on and explain their findings from a physiological standpoint, that is, how are the findings explained. This has much to do with physiologic differences of the thoracic cage between men and women and how pregnancy affects these mechanical properties during pregnancy and with repeated pregnancies. ***This would add a unique aspect to their discussion because there is not much information on the effects of multiparity on respiratory mechanics.RESPONSEWe have taken into account all your suggestions.CORRECTIVE ACTION.In order to improve the scientific quality of our paper, we have added a new table (S2 File) which exposes the main results of the seven studies including healthy females and evaluating the effects of parity on lung function data. We hope that this table will facilitate the interpretation of our data taking into account those of the literature.*We have avoided redundancy between text and tables.**In the revised version, we tried to explain our findings from a physiological standpoint. For that reason, we have added the following sentence (L381-428) related to how pregnancy affects the mechanical properties during pregnancy and with repeated pregnancies. “How to explain the impacts of parity on LFD? During healthy pregnancy, respiratory function is affected through both biochemical and mechanical pathways . Throughout gravidity, spirometry remains within normal ranges . Conversely, lung volumes endure for most variations: ERV progressively declines during the second half of gestation since RV decreases [50-52]. TGV then diminishes while IC rises in the same degree in order to conserve stable TLC [50-52]. Bronchial resistance rises whereas respiratory conductance decreases during gestation [50-52]. Total pulmonary and airways resistances have a tendency to decline in late gestation as a result of hormonally induced relaxation of tracheobronchial tree smooth muscles [51]. What happens with increasing parity? With increasing parity, the rise in TGV, RV and TLC (Tables 1 and 2), and therefore the tendency towards lung-hyperinflation, can be interpreted as an aging index of the ventilatory mechanics, or as an indirect sign towards an OVD and/or an expiratory muscle weakness . The tendency towards lung-hyperinflation can be explained by at least the four following hypothesizes:1) Anatomical changes: during gestation, the progressive increase of the uterus volume is the main reason for lung volume and chest wall changes  . The diaphragm elevation induced two phenomena: i) earlier closure of the lower airways with consequent reduction of TGV and ERV; ii) shorter chest height, but increase of the other thoracic dimensions in order to maintain constant TLC [50-52]. Gestation is also accompanied by changes in the mucosa of the upper and lower airways with the appearance of inflammatory phenomena . Thus, the effects of these changes, can accumulate with repeated gestations. Chest circumference may increase and hypotrophy of the respiratory muscles may develop. This will explain the decline of the maximal inspiratory pressure with high parity (S2 File) [56]. 2) Hormonal changes: during gestation, the physiological adaptation of hormonal  profiles is the foremost cause of ventilatory changes in respiratory function . Progesterone modifies the airways’ smooth muscle tone inducing a bronchodilator effect . Estrogen upsurges the number and the sensitivity of progesterone receptors within several nervous areas  . Prostaglandin F2α rises airway resistance by bronchial smooth muscle constriction, whereas a bronchodilator effect can be a consequence of prostaglandins E1 and E2 [51]. The aforementioned hormonal changes are related to LFD variations . With repeated gestations, it can be speculated that hormonal changes persist and accumulate. During the ageing process, aging-induced hormonal changes can modify LFD [58] . Since gestation is experienced as a stressful situation, hormonal changes can increase in multiparous females. 3) Biochemical changes: the natural damage of elastin with age, contributing to the LFD’ decline, is less accelerated in females with a moderate deficiency in protease inhibitor and having a high parity [60]. This has been attributed to an improvement in elastin turnover in these females with high parity [60]. This finding has not been proven in females with normal protease inhibitor phenotype and high parity [60]. 4) Bronchial hyperreactivity: with gestation, there is a decrease in bronchodilator factors (β2-adrenergic receptors and adenylyl-cyclase activity) in favor of an increase in bronchoconstrictor ones (prostaglandin F2α and cyclic guanosine monophosphatectively) [55]. These effects can accumulate with repeated gestations and partially explain the tendency towards lung-hyperinflation.”***We hope that the actual “Discussion” section is acceptable.REMARK N°3nAgain, these r-values should be squared to determine if they are truly clinically significant – many of the LFD changes will turn out to be not significant or relevant to parity.RESPONSEAgree with your suggestion.CORRECTIVE ACTIONAs previously highlighted, all needed changes were applied (please see our answer to your Remark 9b).REMARK N°3oLast page, last para.: This brief paragraph should be greatly expanded to explain the effects of multiparity on respiratory function , particularly in regards to the lung hyperinflation – how is this event linked to changes in the thoracic cage? In fact, this expanded discussion should nearly completely replace the bloated epidemiologic data from different countries listed by the authors. Pregnancy likely affects the fundamental changes that occur in the respiratory system in a common way, with only subtle differences amongst ethnic/national groups.RESPONSETotally agree with your suggestion.CORRECTIVE ACTION*The old short paragraph was greatly expanded to explain the effects of multiparity on respiratory function.**The paragraph aiming to answer the following question “How to explain the impacts of parity on LFD?” was moved to the main manuscript (L381-428).REMARK N°3pIn short, the information provided here is interesting, and relatively new and should be reported, but in a greatly revised form. It can be presented in a cleaner, more concise manner with greater emphasis placed on the physiologic explanations rather than just reporting epidemiologic data from different countries. The latter aspect can be considerably shortened. Finally, the clinical relevance of statistically significant correlations should be re-considered in a clinically relevant manner.RESPONSEAll your suggestions were accepted and applied.CORRECTIVE ACTION*The paper was deeply revised.**We tried to shorten our paper.***Physiologic explanations are advanced in the main paper.****The clinical relevance of statistically significant correlations should were re-considered in a clinically relevant manner.AttachmentResponses to reviewers.docxSubmitted filename: Click here for additional data file. 29 Oct 2019The impacts of parity on lung function data (LFD) of healthy females aged 40 years and more issued from an upper middle income country (Algeria): a comparative studyPONE-D-19-16514R1Dear Dr. Ben Saad,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Koustubh Panda, M. Tech., Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have made the recommended changes and the manuscript is substantially improved compared to the prior version.Reviewer #2: Authors have addressed the main issues. Much improved manuscript.Much of the material in the appendix has been appropriately transferred to the main text.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  of healthy females aged 40 years and more issued from an upper middle income country (Algeria): a comparative study Dear Dr. Ben Saad:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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Aspergillus fischeri is a common food spoilage fungus and a close relative of the opportunistic human pathogen Aspergillus fumigatus. Here, we sequenced the genomes of two isolates of A. fischeri to build resources for comparative genomics and to aid in differentiation between A. fischeri subspecies. Aspergillus fischeri is a common food spoilage fungus and a close relative of the opportunistic human pathogen Aspergillus fumigatus. Here, we sequenced the genomes of two isolates of A. fischeri to build resources for comparative genomics and to aid in differentiation between A. fischeri subspecies. Aspergillus fischeri is a filamentous fungus that is naturally found in soil and other decaying organic matter (––Aspergillus fumigatus is a closely related species but is responsible for hundreds of thousands of infections and deaths each year (A. fischeri and A. fumigatus diverged as recently as ∼4 million years ago (mya)   and shargo (mya) –12. To dequenced –17, whilequenced .A. fischeri IBT 3003 and IBT 3007, which are part of a collection in the Department of Biotechnology at Technical University of Denmark  and were originally isolated from soil in Denmark (6 spores were inoculated onto potato dextrose agar (PDA) plates at 37°C for 72 hours. DNA was isolated directly from spores using the MasterPure yeast DNA purification kit following the manufacturer’s instructions, with several minor modifications.We sequenced the genomes of  Denmark . For eachttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove residual adaptor sequences and trim reads at low-quality sites using the parameters “–paired,” “–stringency 1,” “–quality 30,” and “–length 50.” The preassembly-improved data sets consisted of 14,619,651 and 15,944,684 read pairs for A. fischeri IBT 3003 and IBT 3007, respectively. These data sets were error corrected and assembled using SPAdes (version 3.9.1) with the “–careful” parameter and k-mer sizes of 31, 43, 73, and 93  region from the A. fischeri IBT 3003 and IBT 3007 genomes, and they were subjected to a BLAST search against the NCBI nonredundant database  was used to evaluate the completeness of the genome assemblies, using the “ascomycota_odb9” gene set  with “–with-quality” and “–pair-by-offset” options to remove potential PCR duplicates . Next, wdatabase . Both IT1 genome . We then1 genome . A. fiscgene set . Totals A. fischeri IBT 3003 and IBT 3007 have been deposited in GenBank under the accession numbers VWYB00000000 and VWYA00000000, respectively. Raw Illumina data have been deposited to the NCBI Sequence Read Archive under the project accession number PRJNA564742.The whole-genome shotgun projects for


E-cigarettes often contain nicotine without the most harmful constituents of tobacco smoke.This study aims to assess prevalence and correlates of accurately perceiving e-cigarettes as less harmful than cigarettes and that none or a small amount of the harm from smoking comes from nicotine.Cross-sectional survey of 2,103 11–18-year-olds in Great Britain in 2016. Prevalence of e-cigarette and nicotine harm perceptions were calculated. Logistic regressions assessed associations between accurate e-cigarette and nicotine harm perceptions and smoking, e-cigarette use, gender, age, region, social grade, family smoking, family e-cigarette use, smoking friends, public approval of smoking, and public approval of e-cigarettes. Associations between accurate e-cigarette and nicotine harm perceptions were also assessed.Most (63.4%) accurate e-cigarette harm perceptions were higher among those aged 16+ (OR = 1.89 [95%CI = 1.45–2.47]), 14–15 (OR = 1.29 [1.00–1.65]), who tried/used an e-cigarette sometimes (OR = 1.51 [1.03–2.21]), with family e-cigarette use (OR = 2.11 [1.46–3.04]), who perceived public disapproval of smoking (OR = 2.11 [1.18–3.77]) and approval of e-cigarettes (OR = 2.44 [1.73–3.45]), and with accurate nicotine harm perceptions (OR = 2.05 [1.28–3.28]). Accurate nicotine harm perceptions were higher among those aged 16+ (OR = 2.60 [1.62–4.16]), from North England (OR = 1.87 [1.02–3.43]) and Wales/Scotland (OR = 2.61 [1.35–5.03]) vs. London, with family smoking (OR = 1.59 [1.05–2.42]), and with accurate e-cigarette harm perceptions (OR = 2.12 [1.32–3.41]).Many young people have inaccurate harm perceptions of e-cigarettes and nicotine. Accurate e-cigarette and nicotine harm perceptions were associated with one another. E-cigarette use was associated with accurate e-cigarette but not nicotine harm perceptions; smoking was not associated with either. WOver the past decade, there have been many studies assessing the relative harm perceptions of e-cigarettes compared to cigarettes. Depending on the sample, year of data collection, and country, the proportion who accurately perceive that e-cigarettes are less harmful than cigarettes varies between 17% and 82% . In adulHarm perceptions of nicotine are often also incorrect. Compared with tobacco, the harms of sustained nicotine use are negligible, and there is little evidence that nicotine increases cancer risk . HoweverThe proportion of inaccurate harm perceptions of e-cigarettes and nicotine is concerning, as it may limit the proportion of smokers willing to try or use a less harmful product which may help them quit. Indeed, adult smokers and ex-smokers who accurately perceive e-cigarettes as less harmful than smoking are more likely to try or currently use an e-cigarette than those who do not hold this perception . HoweverEvidence for associations between harm perceptions of nicotine (or NRT) and using nicotine products is mixed. In addition to assessing associations between accurate harm perceptions and use of both tobacco cigarettes and e-cigarettes, it is also important to identify potential demographic and social correlates of accurate harm perceptions. It is plausible that exposure to smoking and e-cigarettes by family and friends and perceived approval of these products will influence harm perceptions, particularly among youth, whose smoking and e-cigarette uptake has been found to be influenced by some of these social factors , 2014.To our knowledge, there has been no published data on associations between harm perceptions of e-cigarettes and harm perceptions of nicotine among young people. As e-cigarettes often, although not always, contain nicotine, it should be expected that harm perceptions of both would be correlated.Given this, this study has the following two aims: (1) to assess the prevalence of harm perceptions of (a) e-cigarettes relative to cigarettes and (b) nicotine in relation to smoking among young people in Great Britain and (2) to assess the correlates of accurate perceptions that (a) e-cigarettes are less harmful than cigarettes and (b) none or a small amount of the harm from smoking comes from nicotine among young people in Great Britain.22.1https://www.mrs.org.uk/pdf/mrs%20code%20of%20conduct%202014.pdf). Respondents consent to completing surveys in return for a modest financial incentive ; respondents age 11–15 also required consent from their parents or legal guardians.Data were drawn from the cross-sectional 2016 Action on Smoking and Health (ASH) Smokefree Great Britain Youth survey conducted between 11th March and 10th April 2016. This survey is commissioned annually by ASH and is drawn from an online panel maintained by YouGov PLC. Full details of YouGov PLC’s online panel are described in 2.2The sample was designed to be nationally representative in terms of age, gender, and region. The survey was completed by 2,331 respondents age 11–18 inclusive. Respondents who had never heard of e-cigarettes (n = 159), did not report smoking status (n = 32) or e-cigarette status (n = 6), or had missing data for social grade (n = 54) were excluded from the analyses (9.8%). This left 2103 respondents (90.2%) in the study sample.2.3All measures included in this study, item wording, response options, and coding are listed below.2.3.1Relative harm perception of e-cigarettes. Participants were asked: “Compared to cigarettes, do you think e-cigarettes are more or less harmful to the person using them, or is there no difference?” (a) “Less harmful”, (b) “About the same”, (c) “More harmful”, or (d) “Don’t know”. For analysis of correlates of accurate harm perceptions, responses were dichotomised as less harmful (accurate perception) (a) vs. otherwise (b–d).Harm perception of nicotine. Participants were asked: “According to what you know or believe, how much of the harm from smoking cigarettes comes from nicotine?” (a) “None or very small”, (b) “Some but well under half the risk”, (c) “Around half the risk”, (d) “Much more than half the risk”, (e) “Nearly all the risk”, or (f) “Don’t know”. For analysis of correlates of accurate harm perceptions, responses were dichotomized as none or very small (accurate perception) (a) vs. otherwise (b–f). This measure was adapted from 2.3.2Smoking status. Participants identified “Which ONE of the following BEST applies to you?”: (a) “I have never smoked cigarettes, not even a puff or two”, (b) “I have only ever tried smoking cigarettes once”, (c) “I used to smoke sometimes but I never smoke cigarettes now” (d) “I sometimes smoke cigarettes now but less than one a week”, (e) “I usually smoke between one and six cigarettes a week”, (f) “I usually smoke more than six cigarettes a week”, or (g) “Don’t want to say”. For analysis, responses were coded as never (a), tried (b), ex-smoker (c), sometimes (d) and at least weekly (e–f); (g) was excluded.E-cigarette status. Participants identified “Which ONE of the following is closest to describing your experience of e-cigarettes?” (a) “I have never used an e-cigarette”, (b) “I have only tried an e-cigarette once or twice”, (c) “I use e-cigarettes sometimes, but no more than once a month”, (d) “I use e-cigarettes more than once a month, but less than once a week”, (e) “I use e-cigarettes more than once a week but not every day”, (f) “I use e-cigarettes every day”, or (g) “Don’t want to say”. For analysis, responses were coded as never (a), tried or use sometimes (b–c), and at least monthly (d–f); (g) was excluded.2.3.3Family smoking. Participants were asked “Who in your family, if anyone, smokes tobacco cigarettes at the moment? Please tick all that apply” (a) Mother ”, (b) “Father ”, or (c) “Brother or sister”. For analysis, respondents were coded as having at least one family member who smokes if they selected any of (a) through (c).Family e-cigarette use. Participants were asked “And who in your family, if anyone, uses e-cigarettes at the moment? Please tick all that apply” (a) Mother ”, (b) “Father ”, or (c) “Brother or sister”. For analysis, respondents were coded as having at least one family member who uses an e-cigarette if they selected any of (a) through (c).Number of smoking friends. Participants were asked “Please think of the three friends you spend most time with. How many of them smoke tobacco cigarettes on a regular basis?” “0 (none of them)” “1”, “2”, “3 ”, “Don’t know”, or “Don’t want to say”. For analysis, responses were coded as “0 (none of them)”, “1”, “2”, “3 ” and don’t know/refused .Perceived public approval of smoking. Participants were asked “In your opinion, do people in general approve or disapprove of… People smoking tobacco cigarettes?” (a) “Strongly approve”, (b) “Approve, (c) Neither approve nor disapprove, (d) “Disapprove”, (e) “Strongly disapprove”, or (f) “Don’t know”. For analysis, responses were coded as approve (a–b), neither (c), disapprove (d–e), and don’t know (f).Perceived public approval of e-cigarettes. Participants were asked: “In your opinion, do people in general approve or disapprove of… People using e-cigarettes or vaping devices?” (a) “Strongly approve”, (b) “Approve, (c) Neither approve nor disapprove, (d) “Disapprove”, (e) “Strongly disapprove”, or (f) “Don’t know”. For analysis, responses were coded as approve (a–b), neither (c), disapprove (d–e), and don’t know (f).2.3.4Gender was recorded as male vs. female. Age was coded as 11–13 vs. 14–15 vs. 16–18. Participants’ region in Great Britain was coded as North England  vs. South England  vs. London vs. Wales/Scotland. Social grade was coded as ABC1  vs. C2DE . Social grade was based on the occupation of the chief income earner in the household and was asked of the parents of those respondents age 11–15 and directly of those respondents age 16–18.2.4All data were weighted to be representative of age, gender, and region using the 2015 Office for National Statistics census data. All data reported, including sample characteristics, are weighted unless otherwise stated. All analyses used STATA 15.0 .To assess the prevalence of harm perceptions of e-cigarettes relative to cigarettes (aim 1a) and nicotine (aim 1b), prevalence estimates and 95% confidence intervals were calculated.To assess correlates of accurate perceptions that e-cigarettes are less harmful than cigarettes (aim 2a), unadjusted and adjusted logistic regression analyses were used to explore the association between accurately perceiving e-cigarettes to be less harmful than cigarettes (vs. otherwise) and smoking status, e-cigarette status, demographics , social norms , and accurately perceiving that none or a very small amount of the harm from smoking comes from nicotine.To assess correlates of accurate perceptions that none or a small amount of the harm from smoking comes from nicotine (aim 2b), unadjusted and adjusted logistic regression analyses were used to explore the association between accurately perceiving none or a very small amount of harm from smoking cigarettes comes from nicotine (vs. otherwise) and smoking status, e-cigarette status, demographics and social norms (as stated previously), and accurately perceiving that e-cigarettes are less harmful than smoking.33.1Approximately half of respondents were male (51.5%), the majority were from higher social grades ABC1 (68%), and 35.2% were aged 11–13 years, 25.6% aged 14–15 years, and aged 39.2% 16–18 years. Just under half (41.9%) were from North England, 33.5% from South England, 10.9% from London, and 13.8% from Wales/Scotland. The majority were never smokers (80.7%) and never e-cigarette users (87.1%).3.2Almost two thirds  of respondents accurately perceived that e-cigarettes were less harmful than cigarettes, while 488  perceived they were equally as harmful, 56  perceived they were more harmful, and 228  didn’t know.3.3Only a small proportion  of respondents accurately perceived that none or a very small amount of the harm from smoking comes from nicotine, while corresponding values were 385  for “some but well under half of the harm”, 420  for “half of the harm”, 377  for “much more than half of the harm”, 408  for “nearly all of the harm”, and 330  for “don’t know”.3.4In both adjusted and unadjusted analyses, respondents had higher odds of accurately perceiving that e-cigarettes are less harmful than cigarettes if they tried or sometimes use e-cigarettes, were older, had at least one family member who used e-cigarettes, had no smoking friends (vs. responding don’t know or refusing to say), perceive that the public approve or neither approves nor disapproves of e-cigarettes, and accurately perceive that none or a very small amount of the harm from smoking comes from nicotine . In adju3.5In both adjusted and unadjusted analyses, respondents had higher odds of accurately perceiving that none or a very small amount of the harm from smoking comes from nicotine if they were older, from Wales/Scotland compared to London, had at least one family member who smokes, and accurately perceived that e-cigarettes are less harmful than smoking . In adju4In this nationally representative sample of young people in Great Britain, as previously reported , just unThe finding that the majority of young people accurately perceive e-cigarettes as less harmful than cigarettes, yet many still have inaccurate harm perceptions, is similar to some other studies of adults in Great Britain  and the The low prevalence rates of accurately perceiving that none or a very small amount, or even some but under half, of the harm from smoking comes from nicotine is a novel finding. A previous study among adult daily smokers from Sweden and Norway found higher rates of perceiving that none or a very small part (15%) or a relatively small part (31%) of the health risks from smoking come from nicotine , yet theThe higher odds of having accurate e-cigarette harm perceptions in young people who used e-cigarettes less than monthly (vs. never users) is unsurprising and consistent with previous studies in adults  and younUnlike e-cigarette harm perceptions, there was no association between accurate nicotine harm perceptions and e-cigarette use. Therefore, young people may be experimenting with e-cigarettes for reasons unrelated to harm perceptions of nicotine. This could be because not all e-cigarettes contain nicotine or because some individuals do not fully understand the role of nicotine in some e-cigarette use. However, harm perceptions of e-cigarettes and nicotine were positively associated with one another. Qualitative research and surveys including questions relating to these issues and the nicotine content in e-cigarettes may advance understanding of these findings.Neither harm perceptions of e-cigarettes nor nicotine were associated with smoking status. This is contrary to previous studies in youth  and adulIn terms of the demographic correlates, respondents who were younger had higher odds of being misinformed about harm perceptions of nicotine and e-cigarettes consistent with some previous studies . There wIn terms of the social correlates, family use of e-cigarettes was associated with accurate harm perceptions of e-cigarettes. It is possible that some family members who use e-cigarettes were former smokers who overtly acknowledge the relative safety of e-cigarettes and benefits of switching. However, it may suggest that family e-cigarette use could increase the likelihood of youth e-cigarette use if perceptions translate to use, which would be of concern if youth were not smokers. On the other hand, exposure to family smoking was associated with accurate harm perceptions of nicotine (but not e-cigarettes). Number of smoking friends was not associated with harm perceptions of e-cigarettes or nicotine except responding “don’t know” or refusing to answer, which was associated with less accurate harm perceptions of e-cigarettes. Future studies could explore whether friends’ e-cigarette use influences harm perceptions.This was the first study to explore the associations between public approval of e-cigarettes and smoking and harm perceptions of e-cigarettes and nicotine. Perceiving that the public approved of e-cigarettes yet disapproved of smoking was associated with accurate harm perceptions of e-cigarettes. However, public approval of these products was not associated with nicotine harm perceptions except responding “don’t know” or refusing to answer.While this study provides important insight into the harm perceptions of e-cigarettes and nicotine in young people and variables associated with them, the findings must be considered in light of several limitations. First, data are cross-sectional, and therefore it is not possible to infer causality regarding the associations between harm perceptions and use of e-cigarettes/nicotine and product use. Specifically, it cannot be inferred whether young people with accurate harm perceptions of e-cigarettes and nicotine will subsequently try or be tempted to try e-cigarettes, tobacco cigarettes, and other nicotine products. Second, it is not possible to say whether these results generalise to other countries, and the generalisability of the findings to Great Britain may be limited as 10% of the sample was excluded due to having not heard of e-cigarettes or having missing data on key variables. Third, the data are from one time point in March-April 2016, and therefore caution must be exercised if attempting to generalise these findings over time, as harm perceptions are likely to be changing with the continued emergence of research, reports and media stories, and implementation of new laws and regulations . Fourth,Despite these limitations, this study has important strengths. It is the first to assess prevalence rates of the harm perception of nicotine and the first to explore correlates of harm perceptions of e-cigarettes and nicotine among young people in Great Britain. Further, the sample was drawn from the general population in Great Britain using a quota sampling approach and subsequently weighted to enhance representativeness.In conclusion, many young people in Great Britain have inaccurate harm perceptions of e-cigarettes and nicotine. E-cigarette use was associated with accurate harm perceptions of e-cigarettes but not nicotine, while smoking was not associated with either harm perception. Greater understanding of how these harm perceptions influence use of and transitions between e-cigarettes, tobacco cigarettes, and other nicotine products among both smokers and never smokers is needed. While some demographic groups may be more vulnerable to misinformation, specifically younger individuals, continued surveillance of harm perception in addition to nuanced information about e-cigarettes, nicotine, and smoking is essential for all.HC and DA designed the survey, with input from AM, LB and SH. KE led the data analysis and write-up of the manuscript, with significant input from SH, LB and AM. All authors contributed to revisions of the manuscript and approved its final version.All authors declare no conflicts of interest.British Heart Foundation and Cancer Research UK core-funding of ASH. This study was conducted by YouGov PLC. KE, LB, SH, and AM are members of the UK Centre for Tobacco and Alcohol Studies (UKCTAS), which receives funding from The British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, and the Department of Health under the auspices of the UK Clinical Research Collaboration (MR/K023195/1). KE’s PhD is also funded by UKCTAS. LB is funded by a Cancer Research UK/BUPA Foundation Cancer Prevention Fellowship (C52999/A19748). HC and DA are employed by ASH which receives funding from Cancer Research UK, British Heart Foundation, and the Department of Health. Thanks are given to the UK Public Health Research Consortium (PHPEHF50/13) for funding the development of some of the covariates included in this study.This study was led by Action on Smoking and Health (ASH) and was funded by the