Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions . (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in  Aspergillus sydowii is mainly known as a pathogen of corals, which causes tissue lesions and darkening by melanization  aTo describe the morphology of the strain, the fungus was grown at 25 °C on malt extract agar (MEA), Czapek yeast extract agar (CYA), and creatine-sucrose agar (CREA) for 7 days. Additionally, the strain was grown at 30 and 37 °C on CYA and 25 °C on saline CYA (CYAS) with 5% NaCl. Colony diameter, color, texture, production of soluble pigments, and exudates were evaluated in all culture conditions. Conidia and conidiophores were visualized for colonies grown at 25 °C on MEA.For RNA extraction, the strain was cultivated on a semi-solid fermentation of autoclaved (121 °C for 15 min) wheat straw. Three plugs of fresh mycelium grown on PDA without NaCl were inoculated onto 3.0 g of wheat straw soaked with thirty milliliters of Voegel’s medium (without NaCl (No NaCl) and with 0.5 M NaCl or 2.0 M NaCl) in 25.0-cm petri dishes and cultured for 7 days at 28 °C without agitation. For the relative quantification of selected genes by qPCR, an additional group cultured with 1.0 M NaCl in the medium was also considered. The mycelium was collected by adding 15 mL of diethyl-pyrocarbonate (DEPC)-treated water to recover the biomass from the petri dishes and vortexed in sterile conical tubes. This procedure was repeated three times, and the resulting washing liquid was centrifuged to obtain a pellet of cells. Triplicate cultures were obtained for RNA extraction and transcriptome profiling.rpb2), β-tubulin gene (benA), and calmodulin gene (cam) were used as recommended for classification of Aspergillus species [Genomic DNA was extracted from PDA-grown mycelium using standard phenol:chloroform procedure. Primers for internal transcribed spacer (ITS) rDNA, RNA polymerase II second-largest subunit gene  (http://tree.bio.ed.ac.uk/software/figtree/). Further image improvement was performed in CorelDraw Graphics Suite X8 .First, the sequences of ITS,  species  were retutgroups . Multiplutgroups . Maximumml-sms/) . One thoSamples were ground with liquid nitrogen, and RNA purification was performed by the Tri-Reagent method . Libraries were constructed with the TruSeq Stranded mRNA HT kit , and sequencing was performed by the Mass Sequencing Unit of the Biotechnology Institute  with a NextSeq 500 Sequencer  using 150 cycles of pair-end readings.de novo assembly was performed with Trinity [A. sydowii genome for quality control purposes, which rendered a 90.4% overall similarity and approximately 73% coverage. Functional annotation of transcript sequences was achieved by BLAST against nr NCBI RefSeq and UniProt databases using Trinotate software [http://www.tcdb.org) was used to refine the annotations of transcripts belonging to transporter encoding genes of A. sydowii, and substrate specificity of DE transcripts was predicted using the TrSSP tool (http://bioinfo.noble.org/TrSSP/).Sequence reads were filtered/trimmed by quality using FASTQC/Trimmomatic (cutoff: Q20), and  Trinity . The asssoftware . Pfam, Gsoftware ), were ahttp://www.sanger.ac.uk/science/tools/smalt-0). With this alignment, expression levels were quantified using Samtools [The mapping of reads to the assembled transcriptome was done by SMALT  were filtered, aiming to increase the statistical strength of the prediction of differentially expressed transcripts . As showExpression data were normalized using the RUVseq algorithm  for the https://bioconductor.org/). Pathway enrichment analysis was done using the BlastKOALA tool from KEGG database.Gene ontology enrichment was determined by the over-representation analysis (ORA) method with a Fisher’s statistical test using the topGO package (sarA and cox5) were selected according to a previous standardization for Aspergillus niger [sih1, sih2, sih4, and AsHog1 genes was calculated as described by Pfaffl [For validation of RNA sequencing results, 1 µg of total RNA isolated as previously described from semi-solid wheat straw fermentation was treated with DNase I  and used for cDNA synthesis with the RevertAid First-Strand Synthesis kit  using oligo (dT) nucleotides. Two-step quantitative PCR (qPCR) reactions were carried out in a rotor-gene apparatus . Each reaction contained 5 µL of QuantiNova SYBR Green PCR MasterMix and 1 µL of 1:8 dilution of cDNA as the template . The primers used for qPCR, their final concentration on the assay, and the annealing/extension temperature for each primer pair are listed in us niger . Relativy Pfaffl  using tht-test.Fungal tissues were embedded in agarose gel to facilitate fixation procedures, as described elsewhere . BrieflyA. sydowii, the fungus was cultured for 7 days at 150 rpm and 28 °C in 250 mL flasks containing 100 mL of Vogel’s medium (without NaCl (No NaCl) and with 0.5 M NaCl or 2.0 M NaCl) supplemented with 2% glucose as the carbon source. The mycelium was collected by filtration through a 40 μm cell strainer and dried in an oven at 60 °C. Compatible solute extraction for HPLC analysis  was performed as previously described with minor modifications [g for 10 min for phase separation. The upper methanol:water phase was transferred to a 15 mL falcon tube and stored at −20 °C. The HPLC analysis was performed in an isocratic system with an AMINEX-HPX87H column  at 50 °C. Fifty microliters of each sample were injected, and 5mM sulfuric acid was used as the mobile phase at a flow rate of 0.8 mL/min. Glycerol and trehalose standards with an initial concentration of 5 mg/mL were used to construct calibration curves. Chromatogram analysis was performed using ChromQuest software version 2.51 .For the determination of glycerol and trehalose in salt-adapted cultures of Sequencing short read data and processed data were deposited to SRA database under accession number GSE139804, BioProject PRJNA587059. All scripts used for the analysis of high-throughput sequencing data are available from the authors upon request.Aspergillus caesiellus by a polyphasic identification based on: (i) molecular markers ; (ii) micromorphological traits of the colonies, such as shape, size, and grouping of conidiophores and hyphae; and (iii) macromorphological aspects, such as exudates and pigmentation. Although none of the listed molecular markers rendered a resolved phylogram, the authors identified the strain as A. caesiellus, primarily based on micromorphology.The strain BMH-0004 was isolated from a sugarcane bagasse fermentation with 2.0 M NaCl and a posterior screening on a solid medium with carboxymethylcellulose at 0.5 M NaCl . This stAspergillus genus, secondary markers are necessary to ascertain species level with higher confidence [benA, cam, and rpb2 phylogenetic markers were also amplified to aid with identification. The reconstructed phylogeny with these markers showed that strain BMH-0004 is closely related to A. sydowii, Aspergillus section Versicolores [A. caesiellus  Thom and Church [Aspergillus identification is in accordance with A. sydowii. Additionally, microscopic parameters, like biseriate aspergilli, spathulate to subclavate vesicles measuring 10–20 μm, and cylindrical short neck phialides, 5–7 × 3 μm, as well as conspicuously spinose conidia of diameter 3–4 μm, are corresponding. The strain BMH-0004 could grow on solid and in liquid media without NaCl, but its optimal growth was detected when salinity ranged between 0.5 M and 1.0 M NaCl. The maximum tolerated salinity was 2.0 M NaCl when grown on glucose (unpublished data) and complex substrates (carboxymethylcellulose or wheat straw) [The morphology of this strain confirmed the characteristics of d Church . It is mA. sydowii transcriptomes of the fungus growing without NaCl or at 2.0 M NaCl  were more similar to each other than to the transcriptome at the optimum salinity (0.5 M NaCl). This suggests that the expression profile under nonoptimal conditions is driven by stress-related responses, which include a global upregulation of central catabolic and secondary metabolism transcripts and substrate-degrading enzymes  and a methyl-sterol monooxygenase at 0.5 M NaCl (logFC 0.5 M NaCl vs No NaCl = 2.29). The overexpression of the former would imply an increase in unsaturated lipids, while the latter is involved in the ergosterol synthesis pathway. The action of these enzymes possibly increases membrane fluidity, which was observed previously in the extremely halotolerant black yeast Hortaea werneckii and the halotolerant black yeast Aureobasidium pullulans [In nt fungi ,46. Whilullulans .A. sydowii, this structure is extensively modified in salinity conditions -glucans forming a rigid hydrophobic core surrounded by cross-linked β(1-3)-glucans [Transcriptional changes of genes associated with construction, deconstruction, and remodeling of the fungal cell wall suggest that, in nditions . The cur-glucans ,48,49. CA. fumigatus, eight CHS (chsA–G) genes have been identified, but their specific biological roles are still unclear, as deletion of many of these genes had no observable effect on growth or chitin content in the cell wall [A.sydowii, at least 11 different transcripts encoding eight chitin synthases genes (chs) were identified, but only one of these was strongly downregulated (logFC 2.0 M NaCl vs No NaCl = −3.18) when growing in the presence of salt. Nonetheless, the cumulative transcription level of all chs genes did not change significantly, suggesting that chitin synthesis is not affected  and chitotriosidase (chit) transcripts levels were upregulated when the fungus grew in 2.0 M NaCl, which may result in an overall decrease of the chitin and chitosan content in the cell wall.The amount of chitin in cell walls is a function of its synthesis and degradation. Chitin synthases (CHS) are membrane proteins that polymerize intracellular UDP-N-acetyl-glucosamine and extrude the chitin polymer to the cell wall space. In ell wall . In A.syaffected A. AdditiA. fumigatus, chsE and chsG deletion mutants displayed substantial changes in growth, hyphal morphology, conidiation, and chitin content, with a decrease in chitin being counterbalanced by an increase in α-glucan [A. sydowii suggested that, in response to salinity, the increase in β-glucans was more pronounced than that of α-glucans (As shown in )-glucan . Interes-glucans B.A. fumigatus is mainly composed of β-glucans and α-glucans. The β -glucan is synthesized as a linear polymer by the FKS1 synthase complex, while the extruded chains can be transferred to extend existent β-glucan or to GPI-anchored proteins that link cell wall polysaccharides to the cell membrane. Four families of GPI-anchored proteins  were found to be common to all fungi [A. sydowii transcriptome, indicating the importance of cross-linkage modifications for maintaining cell wall integrity during osmotic stress, as discussed below.The cell wall of ll fungi ,53. Geneecm33 gene display increased sensitivity to a variety of stress conditions, such as oxidative agents, fungicides, cell wall perturbing agents, and osmotic and ionic imbalances [ecm33 genes in A. sydowii was almost two times greater at 2.0 M NaCl than at the other two tested conditions, which indicates they favor the fungus’ stress tolerance.ECM33 proteins are involved in the correct assembly of the cell wall β-glucan and the mannoprotein layer . Severalbalances ,56. ECM3crh mutants of Saccharomyces cerevisiae, the chitin fraction is completely devoid of linkages to β-glucans [crh genes in Candida albicans increases the elasticity of the cell wall, reducing survival during osmotic shock, while overexpression of one crh gene has an osmoprotective effect [A. sydowii, where a CRH homologue gene (crf) was overexpressed at 2.0 M NaCl (logFC 2.0 M NaCl vs No NaCl = 1.23)  B.A. fumigatus, seven gel genes have been identified, of which only three  are expressed during mycelial growth [gel4 is an essential gene [gel2 mutants have reduced growth, and gel1 deletion has no evident effect on morphology [Neurospora crassa, individual deletions of gel genes do not influence stress sensitivity [A. fumigatus. Noticeably, if the function of GEL proteins is abolished—with a triple mutation in N. crassa [gas1 in S. cerevisiae [A. sydowii, the same balancing effect can be observed. At 2.0 M NaCl, chitin content should be lower due to increased chitin degradation, which is compensated by β-glucan cross-linking as a result of higher GEL expression.Finally, the GEL family of glucanosyltransferases is the most studied among cell wall-associated proteins, and its members are present in a relatively high amounts in fungal cell walls. Their function is to cleave the newly synthesized linear β-glucan and transfer it to another β-glucan molecule, resulting in elongation or shortening of the glucan fibers. GEL proteins also regulate the cross-linking of proteins into the cell wall, therefore allowing the correct assembly of cell wall structures . In A. fl growth ,60. In trphology . In Neursitivity , indicat. crassa  or a sinrevisiae —the chitA. sydowii supported this notion, showing that the cells grown in 2.0 M NaCl exhibit significantly thicker cell walls compared to the other two investigated conditions  generate a reduction in cell wall elasticity that allows resistance to external insults. Ultrastructural analysis of nditions C. Furthenditions D.One of the novel halophilic strategies described in fungi is the production of hydrophobins (HFBs), which have been observed in both basidiomycete and ascomycete model fungi when growing in high salinity. HFBs are small extracellular proteins produced exclusively by filamentous fungi, which means that even yeasts do not contain genes for these proteins. HFBs self-assemble at hydrophobic-hydrophilic interfaces and have been associated with the morphogenesis of aerial hyphae and the adhesion of hyphae to hydrophobic surfaces . The abuWallemia ichthyophaga, there are 26 HFB genes, more than twice the number found in related species of the genus. At the low-salinity growth limit of this species (10% NaCl), eight HFB genes are overexpressed, while four are overexpressed at high salt concentrations (30% NaCl) [Individual species of filamentous fungi usually contain between two and seven HFB genes . In the 0% NaCl) . Another0% NaCl) . This su0% NaCl) , but thiAspergillus, the estimated number of HFB per species is between six and 10, with an average of nine [A. sydowii transcriptome, we identified four HFBs (sih1-4), fewer than in some other members of the genus. Of these, only three  were completely represented in the sequenced genome of A. sydowii. In the genomic locus corresponding to the sih4 transcript, there is a truncated version of the gene that codes for a protein product with only four cysteine residues. As this transcript was highly expressed when the fungus was grown without NaCl, we cloned the sih4 coding sequence from cDNA and confirmed that the transcript corresponds to a complete HFB gene (data not shown). This gene might have been lost (or repurposed) during the migration of A. sydowii from the terrestrial to the marine habitat, which has been previously proposed as a mechanism of evolution of HFB genes in the ascomycete family of Hypocreales [Particularly in the genus  of nine . In the ocreales . It alsoocreales .A. sydowii hydrophobins do not have a high proportion of acidic residues, as observed for their orthologues in the halophilic W. ichthyophaga, and their pI values do not distinguish them from their counterparts in nonhalophilic fungi  indicated that only  to zero E. Relatinditions F. On thent years , but thent years , thus prH. werneckii and W. ichthyophaga, glycerol is the most abundant osmolyte, but also, erythritol, arabitol, and mannitol can be found in lesser amounts [A. niger, glycerol and erythritol accumulate in young mycelium, whereas mannitol and erythritol are the signature compatible solutes in old mycelium [A. niger and A. nidulans, trehalose and mannitol have been found [Among the most crucial adaptations to salinity in fungi is an increased concentration of intracellular compatible solutes. For example, in  amounts ,72. Simimycelium ,74. In cen found ,76.A. sydowii BMH-0004 strain, some transcripts associated with the metabolism of osmolytes were differentially regulated in response to osmotic stress. Noteworthy was the expression of genes involved in trehalose synthesis, where α-trehalose phosphate synthase (TpsA) was expressed under both hypo- and hyperosmotic stress , encoding the rate-limiting enzyme in the pathway of glycerol synthesis. At the same time, the transcription of the genes encoding the STL1 glycerol:H+ transporters, which facilitate the import of glycerol into the cells, also increased at 2.0 M NaCl. Glycerol was accumulated only at 2.0 M NaCl and not at the optimal salinity for growth  of the glycerol-3-phosphate dehydrogenase gene . The activation of the HOG pathway induces the accumulation of glycerol, arrest of the cell cycle, and reorganization in the actin cytoskeleton, as well as changes in cell wall dynamics ,81. In tic shock F.+) [A. sydowii and A. versicolor, and the halotolerant relatives A. nidulans, A. niger, and A. fumigatus ; MFS members (Pfam 07690 and 00083); and cation/transporter ATPase exchangers  showed striking differences  revealed a higher number of genes encoding APC members, which could have a role in ensuring a rapid uptake of certain amino acids under hypersaline conditions. Osmoregulation at increased salinity in prokaryotes often involves accumulation of amino acids, such as proline and betaines, which could be also the case in halophile fungi growing in rich substrates. The contribution of the APC transporters to halotolerance remains underexplored in filamentous fungi.Although not universally, some fungal halophilic strains have a higher number of transport systems—when compared with mesophilic relatives – to regulate the homeostasis of alkali cation levels, as well as the fluxes across the plasma membrane, to eliminate toxic ions such as sodium (Na+) ,82,83. Fumigatus . The halferences . The genA. sydowii was grown at 0.5 M NaCl but not at 2.0 M NaCl , but their expression level mean across all conditions was 1.36 tpm.Gene expression analysis showed an enrichment of transport-related genes when 0 M NaCl . In totalinities . One posena2 gene (ATN2), a sodium P-type ATPase commonly associated with salinity adaptations in the yeast Saccharomyces cerevisiae and other fungi, was downregulated at 0.5 M NaCl when compared to the conditions without salt or 2.0 M NaCl. This gene was found differentially expressed in W. ichthyophaga and H. werneckii under high salinity conditions [A. sydowii, the expression of ena2 is regulated by stress signals rather than high concentrations of salt. ENA P-type ATPase family in fungi are functionally different from animal Na+, K+- ATPases, as they have lower selectivity to cations  [+ concentrations, the ENA P-type ATPase pumps out K+ to maintain the Na+/K+ ratio, whereas the opposite occurs in hypersalinity conditions. Supporting this notion, the K+/H+ antiporter kha1 gene, which presumably extrudes K+ from the cell or into intracellular vesicles [atc3 and atc9 genes are other putative P-type ATPases with the same expression profile as the ena2 gene in A. sydowii, suggesting that the fungus actively transport cations under nonoptimal conditions to maintain the Na+/K+ ratio.The nditions . Our rescations) . In condvesicles ,86, was A. sydowii in three different osmotic conditions, we learned that physiological responses to salinity vary greatly and are not a simple graded effect as the salt concentration of the medium is increased. In fact, in each condition assayed, the responses could be assumed as a distinct physiological state  (sih1 and sih2).By comparing the transcriptomic profiles of al state . When gre (sih4) . Stress A. sydowii, these are not taking place concurrently. The adaptations to ensure osmotic balance and avoid ion toxicity when the fungus grows under optimal salinity conditions are not completely understood, as they do not coincide with the halophile adaptations described in the literature. Further experiments should be conducted to identify and understand these adaptations and the effects of different stress signals on modulating the response. Our results indicate that the interpretation of physiological reactions in extremophiles or extremotolerant microorganisms should be addressed differently, as stress can reshape the physiological outcome. In this sense, the strain A. sydowii BMH-0004 was a useful model to observe these transitions.While most of the mechanisms of adaptation to salinity described to date in eukaryotes are also occurring in


These reactions are site-selective, yielding, respectively, 17,18- or 12,13-dihydroporpholactones. The crystal and molecular features of pyrrolidine-fused and isoxazolidine-fused dihydroporpholactones were unveiled from single-crystal X-ray diffraction studies.The reaction of  Pyrrole-modified porphyrins have attracted considerable attention in recent years. This group of compounds comprises a large diversity of porphyrin analogues where one or more pyrrolic units of the porphyrin macrocycle are replaced by non-pyrrolic units. The synthesis, reactivity, structural features, optical properties, and applications of these compounds have been comprehensively reviewed ,3,4,5,6.meso-tetraarylporphyrins in a two-step procedure  ), 7.32 , 7.59 , 7.77 . 13C NMR : δ 40.9, 47.6, 48.8, 62.0, 62.2, 83.2, 95.7, 109.3, 110.2, 110.6, 112.5, 113.7, 114.1, 122.0, 123.3, 134.7, 135.6, 135.9, 137.0, 138.5, 139.0, 141.2, 143.2, 144.1, 144.8, 145.4, 146.1, 146.6, 147.3, 151.2, 154.1, 157.6, 159.1, 162.2, 164.6. 19F NMR : δ −158.0 to −155.9 , −148.3 to −147.2 , −135.6 to −132.0 . UV-Vis (CHCl3): λmax (log ε) 382 (5.23), 531 (4.38), 572 (4.60), 617 (3.93). MS (ESI): m/z 1050.5 [M + H]+.Adduct 3b: , 1H NMR : δ 2.53–2.60 , 2.84 , 3.46 , 3.58 , 4.45–4.63 ]), 7.06–7.09 , 7.15–7.18 , 7.28–7.30 , 7.59 , 7.77 . 13C NMR : δ 47.0, 48.2, 59.1, 59.7, 59.9, 83.1, 95.7, 109.4, 110.2, 110.5, 112.5, 114.0, 122.0, 123.2, 125.3, 127.7, 128.2, 128.4, 128.8, 129.05, 129.11, 134.8, 135.6, 135.8, 136.8, 138.8, 141.0, 143.2, 144.0, 144.5, 145.2, 146.0, 146.4, 146.8, 147.3, 151.3, 154.1, 157.6, 159.1, 162.3, 164.6. 19F NMR : δ −158.0 to −155.9 , −148.3 to −147.3 , −135.7 to −131.8 . UV-Vis (CHCl3): λmax (log ε) 382 (5.21), 532 (4.34), 572 (4.59), 629 (3.56). MS (ESI): m/z 1126.1 [M + H]+.Adduct 1  in toluene (5 mL), N-substituted hydroxylamine hydrochloride (0.20 mmol), paraformaldehyde , and K2CO3  were added. The mixture was heated at 60 °C under a nitrogen atmosphere for 12 h, and then additional portions of N-substituted hydroxylamine hydrochloride, paraformaldehyde, and K2CO3 were added to the reaction mixture, which was heated for a total of 30 h. The reaction mixture was washed with H2O and then was purified by preparative TLC using dichloromethane/hexane (1:1) as the eluent. The fraction with higher Rf corresponded to unreacted porpholactone (~60%), the following one was the major cycloadduct, and the third one the minor cycloadduct. Isolated yields: 6a = 19% (48% yield based on consumed porpholactone 1), 7a = 8% (19% yield based on consumed porpholactone 1); 6b = 22% (43% yield based on consumed porpholactone 1), 7b = 9% (18% yield based on consumed porpholactone 1).To a solution of porpholactone 6a: 1H NMR : δ −1.53 , −1.19 , 2.57 , 2.86–2.95 , 3.35–3.43 , 5.14–5.19 ]), 6.65 ]), 8.19 , 8.48–8.52 , 8.64 . 13C NMR : δ 44.0, 55.4, 64.0, 85.0, 89.7, 98.7, 102.3; 105.5, 105.8, 110.7, 111.4, 114.5; 122.9, 125.3, 126.8, 127.9, 128.1, 128.2, 128.3, 134.7, 137.5, 138.2, 138.9, 140.9, 141.6, 143.5, 145.1, 147.1, 148.9, 152.6, 162.9, 165.8. 19F NMR : δ −158.2 to −155.7 , −148.3 to −146.7 , −136.0 to −131.1 . UV-vis (CHCl3): λmax (log ε) 404 (5.30), 497 (4.07), 530 (3.86), 625 (3.75), 684 (4.61). MS (ESI): m/z 1052.5 [M + H]+.Adduct 7a: 1H NMR : δ −1.67 , −1.36 , 2.57 , 2.90–2.95 , 3.39–3.44 , 5.21–5.23 ]), 6.66 ]), 8.33 , 8.40 , 8.56 , 8.66 . 19F NMR : δ −158.4 to −155.6 , −148.7 to −146.1 , −136.3 to −131.2 . UV-vis (CHCl3): λmax (log ε) 404 (5.44), 496 (4.20), 530 (3.97), 625 (3.90), 684 (4.83). MS (ESI): m/z 1052.2 [M + H]+.Adduct 6b: 1H NMR : δ −1.68 , −1.31 , 3.25 , 3.81 , 5.24–5.25 , 6.65 ]), 7.00–7.02 , 7.19–7.22 , 8.22 , 8.46 , 8.55 , 8.66 . 13C NMR : δ 44.0, 53.9, 64.6, 86.1, 89.3, 99.6, 101.0, 105.8, 110.7, 112.7, 113.7, 115.3, 122.3, 125.4, 127.3, 128.3, 136.8, 137.5, 138.9, 139.3, 139.4, 141.2, 141.6, 143.3, 143.7, 144.8, 145.2, 146.9, 152.9, 163.0, 165.8. 19F NMR : δ −158.2 to −155.8 , −148.2 to −146.7 , −135.7 to −130.1 . UV-vis (CHCl3): λmax (log ε) 404 (5.92), 496 (4.66), 529 (4.46), 623 (4.35), 646 (4.33), 682 (5.24). MS (ESI): m/z 1128.4 [M + H]+.Adduct 7b: 1H NMR : δ −1.73 , −1.43 , 3.31 , 3.85 , 5.33–5.37 , 6.67 ]), 7.02–7.06 , 7.19–7.23 , 8.30 , 8.42 , 8.57 , 8.68 . 19F NMR : δ −158.3 to 155.5 , −148.3 to −146.1 , −136.3 to −130.0 . UV-vis (CHCl3): λmax (log ε) 404 (5.57), 497 (4.30), 529 (4.06), 626 (3.97), 643 (4.05), 684 (4.87). MS (ESI): m/z 1128.3 [M + H]+.Adduct www.ccdc.cam.ac.uk/data_request/cif, by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.CCDC 1832879, 1832880, and 1985131 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via


In Ghana, home delivery among women in urban areas is relatively low compared to rural areas. However, the few women who deliver at home in urban areas still face enormous risk of infections and death, just like those in rural areas. The present study investigated the factors associated with home delivery among women who live in urban areas in Ghana.Data for this study was obtained from the 2014 Ghana Demographic and Health Survey. We used data of 1,441 women who gave birth in the 5 years preceding the survey and were dwelling in urban areas. By the use of Stata version 14.2, we conducted both descriptive and multivariable logistic regression analyses.We found that 7.9% of women in urban areas in Ghana delivered at home. The study revealed that, compared to women who lived in the Northern region, women who lived in the Brong Ahafo region  were less likely to deliver at home. The likelihood of home delivery was high among women in the poorest wealth quintile , women who professed other religions , and those who had no antenatal care visits . Conversely, the likelihood of home delivery was lower among women who had attained secondary/higher education , compared to those with no formal education.The study identified region of residence, wealth quintile, religion, antenatal care visits, and level of education as factors associated with home delivery among urban residents in Ghana. Therefore, health promotion programs targeted at home delivery need to focus on these factors. We also recommend that a qualitative study should be conducted to investigate the factors responsible for the differences in home delivery in terms of region, as the present study could not do so. Reducing maternal mortality has been one of the greatest public health concerns. In line with this, the United Nations instituted the Millennium Development Goals (MDGs) in 2000 with Goal 5 aimed at reducing maternal mortality by 75% by the year 2015 . In 2015In low- and middle-income countries (LMICs), several significant efforts have been made to reduce maternal deaths through enhanced maternal healthcare services utilization globally –10. HoweA larger percentage of maternal mortalities in Ghana is caused by pregnancy-related issues such as obstetric complications, which result in death during pregnancy, childbirth, or within 42 days after delivery . This imOver the years, the government of Ghana has attempted to improve access to maternal healthcare services. In 2003, the government, for example, introduced the waiver of delivery fees, and by 2005, fees on delivery care were abolished in all the then 10 regions of the country . This waIt is, therefore, important to understand the factors associated with home delivery among urban women in Ghana and provide useful information for interventions aimed at reducing maternal mortality in the country. In Ghana, a few studies have been conducted in this regard. Studies by Ganle et al.  and BoahThe study setting is the Republic of Ghana. The Republic of Ghana is one of the countries in West Africa and has a total land area of 238,533 square kilometres ?” In the GDHS, responses to this question were home, other home, government hospital, government health centre/clinic, government health post/ Community-based Health Planning and Services (CHPS), other public, private hospital/clinic, maternity homes, and others. These responses were dichotomised into health facility delivery = 0 and home delivery = 1. Home delivery referred to deliveries that occurred in respondent’s home and other home. On the other hand, deliveries that occurred at government hospital, government health centre/clinic, government health post/CHPS, other public, private hospital/clinic, maternity homes, and other health facilities were grouped as “health facility delivery.”The study considered twelve explanatory variables. These are age, region, religion, ethnicity, educational level, marital status, wealth status, employment, parity, sex of household head, ANC visits, and decision-making for healthcare. These variables were not determined a priori; instead, they were determined based on parsimony, theoretical relevance, and practical significance with place of delivery –37. The ). The results were presented as adjusted odds ratios, with their corresponding 95% confidence intervals signifying their level of precision. Statistical significance was declared at p<0.05. Sample weight was applied and the survey command (svy) was used to account for the complex sampling design of the survey. We wrote the manuscript following the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) statement.The statistical software Stata version 14.0 was used to process the data. Both bivariate and multivariate analyses were employed in this study and results were tested at 95% confidence interval. Bivariate analysis was conducted to show the proportion of home deliveries across socio-demographic characteristics with their significance levels and chi-square values (χ2). Multivariable analysis (binary logistic regression) was further conducted. Only the variables that showed statistical significance in the bivariate analysis were included in the regression analysis. Before the binary logistic regression analysis, we conducted a multicollinearity test of all the statistically significant variables using the variance inflation factor (VIF), and it showed no evidence of collinearity among the explanatory variables . You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Frank T. SpradleyAcademic EditorPLOS ONEJournal requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  http://learn.aje.com/plos/) for a 15% discount off AJE services. 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We noticed you have some minor occurrence of overlapping text with the following previous publication, which needs to be addressed:https://journals.plos.org/plosone/article/file?id=10.1371%2Fjournal.pone.0220970&type=printable- In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. 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: YesReviewer #3: 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: NoReviewer #3: 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: Background information, the following are missing1. Clear definition of home birth is missing2. What is situation in Ghana, does home birth = unskilled assistance3. How does this study define unskilled birth assistance?4. It must come clearly why urban? The expectation is urban home birth may invite skilled attendant as compared to rural? 5. Are there traditional birth attendants in urban?The paper is about predictors of home birth but authors have to say something on the proportion of home birth vs health facility birth.Reviewer #2: Review report ( Dr. Habtamu Tolera)The manuscript reports the findings regarding “What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey data (PONE-D-20-23864). This kind of study is much relevant in context of LMICs like Ghana. The sample size is adequate and the analyses is really very rigorous and well done. I have found that authors strongly need to rework on the background and methodology sections. They also need to avoid long sentences used across the manuscript. Here below in my report I have tried to review some points or concerns that need authors to further rework on them to improve the power of this manuscript to get published in PLoS One Journal. Authors can also refer to the attached pdf file.Affiliation:Corresponding author's E-mail address is enough. So, remove others’ email addresses from the front page of the manuscriptBackground:• I have found the sentences in the first paragraph of the “Background” section of this study  were too long and lacks clarity. Thus, I advise authors: (1) avoiding long sentences from this section and elsewhere across the manuscript, (2) clarify all so that they convey the correct information for potential readers of the manuscript.• See Page # 3, line 97. If this is so, why do the author(s) interested to urban Ghana than rural Ghana in this study? authors need to give justifications for selection criteria.• See also sentences on page 3, lines 103 through 110. They were not clearly stated for potential international readers, all have been needed to be rewritten.• Page 3, Lines 110-112 said like this, “However, these studies focused more on rural areas, ignoring the fact that a considerable number of women in urban areas still use home delivery service” This needs authors to acknowledge (cite) supporting evidences to say so. So that they may convince the readers.• Finally, your “Background” section needs more detail. I suggest that authors improve the description at "Background" section to provide more justification for their study . I also recommend authors to read and review adequate empirical works on home delivery elsewhere in LMICs to enrich also their "Background" section. Why did you stick to works done in Ghana alone? They need to read vast works beyond urban Ghana and describe the contexts, background pictures  of the topic; empirical/theoretical/methodological gaps observed with regard to the issue/the problem under study as well. Authors again need to state the benefits of this research findings (for program/policy interventions) in the last sentence of the "background" section or somewhere in this section.Methods and materials• I understand that authors analyzed GDHS or survey data collected by government. However, I have found that important methodological subsections/elements or components were missing in this study. So, authors need to add "Study setting", "study design", "Sampling procedure/sample size determination", "Data collection/quality assurance", "Data processing procedures and analyses". They need to customize these methodologies from GDHS "survey they used for this study. These are mandatory to be included under the methodological sections to be published in PLoS ONE.• On Page 3, lines 119 through 121 Under “Data Source” section, it was stated/listed like this, “The survey targets core maternal and child health indicators such as unintended pregnancy, contraceptive use, skilled birth attendance, immunization among under-fives, and intimate partner violence”. Did these variables were captured/considered in the report/ analyses of this study? I have not they found them. If not relevant, please remove it If were not well addressed or if they were inserted by mistake.• Line 133, better if you label it like this, "Variables studied" or “Measurements”• Sentences from Lines 135 through 140, is not clear. The current phrasing makes comprehension difficult. so, have been rewritten.• A sentence from Lines 135-38, were put like this, “The outcome variable employed for this study was “home delivery” which was obtained from the 136 question, “Where did you deliver?” Responses to this question were coded respondent’s home, other home, government hospital, government health centre/clinic, government health post/CHPS, other public, private hospital/clinic, maternity homes, and others”. My concern is that, under which category did authors assign or grouping "maternity home” in this coding of categories of the outcome variable, delivery choice status of study subjects. They need to recheck the codingResults:• Authors need to insert a new column in Tables 1 and 2 to report the frequency and the proportion of both home delivery and Non-home delivery statuses of women. Readers need to get these two values together AOR, CI, P-values, etc.• Finally, some texts and paragraphs need editorial problems and authors need to rewrite with clear and plain languageReviewer #3: This is an informative piece of work.However, it would do better with inclusion of recent studies on home delivery in the introduction and or discussion to make it stronger.Summarize all the key findings at the beginning of the discussion to get the reader a better perspective vs the discussion of results separately in fragmented styleThe conclusion can still be strengthened, made more clearer and indicative.Take care of references to ensure consistency, providing key links for critical documents and when these were accessed.**********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, AttachmentFinal HOME DELIVERY_Vancouver copy.docxSubmitted filename: Click here for additional data file.AttachmentGahan paper PONE-D-20-23864_reviewer.pdfSubmitted filename: Click here for additional data file. 16 Nov 2020AUTHOR’S RESPONSE TO REVIEWSTitle: What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey dataDate: 16/11/2020AUTHOR’S RESPONSE TO REVIEWSDear Editor and Reviewers,We are pleased to resubmit for publication the revised version of “What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey data”. Thank you for giving us the opportunity to revise and resubmit this manuscript. We appreciate the time and constructive feedback provided by the reviewers and the Editor. We have added an additional author Anita Gracious Archer based on her valuable inputs during the revision process. The manuscript has certainly benefited from these insightful suggestions. Overall, the revision process is very productive. We have made every attempt to fully address all the comments in the revised manuscript. period. Based on the comments, we have responded specifically to each suggestion below. RESPONSE TO REVIEWS Reviewer #1: Background information, the following are missing1. Clear definition of home birth is missing2. What is situation in Ghana, does home birth = unskilled assistance3. How does this study define unskilled birth assistance?4. It must come clearly why urban? The expectation is urban home birth may invite skilled attendant as compared to rural? 5. Are there traditional birth attendants in urban?Response: The background has been revised to incorporate all these suggestions (Line 99-104).The paper is about predictors of home birth but authors have to say something on the proportion of home birth vs health facility birth. Response: We have mentioned that “We found that 7.9% of women in urbans areas in Ghana delivered at home” (Line 55).Reviewer #2: Review report ( Dr. Habtamu Tolera)The manuscript reports the findings regarding “What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey data (PONE-D-20-23864). This kind of study is much relevant in context of LMICs like Ghana. The sample size is adequate and the analyses is really very rigorous and well done. I have found that authors strongly need to rework on the background and methodology sections. They also need to avoid long sentences used across the manuscript. Here below in my report I have tried to review some points or concerns that need authors to further rework on them to improve the power of this manuscript to get published in PLoS One Journal. Authors can also refer to the attached pdf file.Response: Thank you for your useful comments. We have considered all of them in the revised manuscript. Affiliation:Corresponding author's E-mail address is enough. So, remove others’ email addresses from the front page of the manuscriptResponse: We have taken out the E-mail addresses of the co-authors and left only that of the corresponding author. Background:• I have found the sentences in the first paragraph of the “Background” section of this study  were too long and lacks clarity. Thus, I advise authors: (1) avoiding long sentences from this section and elsewhere across the manuscript, (2) clarify all so that they convey the correct information for potential readers of the manuscript.Response: We have clarified the information provided in that paragraph (Line 73-84)• See Page # 3, line 97. If this is so, why do the author(s) interested to urban Ghana than rural Ghana in this study? authors need to give justifications for selection criteria.Response: We have revised the background and provided justification for conducting the study using urban women. A recent study and a number of previous studies have focused on rural Ghana despite urban women who deliver at home still going through the same risk of infections and deaths as their rural counterparts (Line 113-120).• See also sentences on page 3, lines 103 through 110. They were not clearly stated for potential international readers, all have been needed to be rewritten.Response: We have revised those sentences (Line 121-130).• Page 3, Lines 110-112 said like this, “However, these studies focused more on rural areas, ignoring the fact that a considerable number of women in urban areas still use home delivery service” This needs authors to acknowledge (cite) supporting evidences to say so. So that they may convince the readers.Response: We have cited sources for the information (Line 130)• Finally, your “Background” section needs more detail. I suggest that authors improve the description at "Background" section to provide more justification for their study . I also recommend authors to read and review adequate empirical works on home delivery elsewhere in LMICs to enrich also their "Background" section. Why did you stick to works done in Ghana alone? They need to read vast works beyond urban Ghana and describe the contexts, background pictures  of the topic; empirical/theoretical/methodological gaps observed with regard to the issue/the problem under study as well. Authors again need to state the benefits of this research findings (for program/policy interventions) in the last sentence of the "background" section or somewhere in this section.Response: We have revised the background section to incorporate all these useful suggestions Methods and materials• I understand that authors analyzed GDHS or survey data collected by government. However, I have found that important methodological subsections/elements or components were missing in this study. So, authors need to add "Study setting", "study design", "Sampling procedure/sample size determination", "Data collection/quality assurance", "Data processing procedures and analyses". They need to customize these methodologies from GDHS "survey they used for this study. These are mandatory to be included under the methodological sections to be published in PLoS ONE.Response: We have considered these useful suggestions under “Materials and methods”.• On Page 3, lines 119 through 121 Under “Data Source” section, it was stated/listed like this, “The survey targets core maternal and child health indicators such as unintended pregnancy, contraceptive use, skilled birth attendance, immunization among under-fives, and intimate partner violence”. Did these variables were captured/considered in the report/ analyses of this study? I have not they found them. If not relevant, please remove it If were not well addressed or if they were inserted by mistake.Response: We have removed these from the paper (Line 160-163). • Line 133, better if you label it like this, "Variables studied" or “Measurements”Response: We have now used variables studied (Line 194).• Sentences from Lines 135 through 140, is not clear. The current phrasing makes comprehension difficult. so, have been rewritten.Response: We have made the paragraph very clear (Line 196-204). • A sentence from Lines 135-38, were put like this, “The outcome variable employed for this study was “home delivery” which was obtained from the 136 question, “Where did you deliver?” Responses to this question were coded respondent’s home, other home, government hospital, government health centre/clinic, government health post/CHPS, other public, private hospital/clinic, maternity homes, and others”. My concern is that, under which category did authors assign or grouping "maternity home” in this coding of categories of the outcome variable, delivery choice status of study subjects. They need to recheck the codingResponse: In line with the definition of home delivery and health facility delivery in the DHS and in previous studies, ‘maternity home’ is considered part of health facility delivery in this study. Results:• Authors need to insert a new column in Tables 1 and 2 to report the frequency and the proportion of both home delivery and Non-home delivery statuses of women. Readers need to get these two values together AOR, CI, P-values, etc.Response: Per our understanding of your suggestion, we have inserted a column in Table 1 to report on the frequency and proportion of home delivery and health facility delivery. We are unsure if you also suggested we insert a column in Table 2 to report AOR, CI and P-values for health facility delivery as this is not appropriate since the outcome of interest in this study was “home delivery” and the AOR, CI and P-values produced in the regression analysis are only for the outcome of interest and not for both “home delivery” and “health facility delivery”. • Finally, some texts and paragraphs need editorial problems and authors need to rewrite with clear and plain languageResponse: We have addressed all these issues. Reviewer #3: This is an informative piece of work.However, it would do better with inclusion of recent studies on home delivery in the introduction and or discussion to make it stronger.Summarize all the key findings at the beginning of the discussion to get the reader a better perspective vs the discussion of results separately in fragmented styleThe conclusion can still be strengthened, made more clearer and indicative.Take care of references to ensure consistency, providing key links for critical documents and when these were accessed.Response: Thanks for your useful comments suggestions. All these issues have been considered in the revised paper  10 Dec 2020PONE-D-20-23864R1What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey dataPLOS ONEDear Dr. Budu,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Carefully proof your manuscript to correct grammar or spelling errors.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jan 24 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Frank T. SpradleyAcademic EditorPLOS ONEReviewers' 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 addressedReviewer #3: 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: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: 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: YesReviewer #3: 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: YesReviewer #3: 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: Authors have addressed the raised comments as required,Minor revisions on the abstract and some typographical errorsReviewer #2: I hope authors did all best. I appreciate your commitments. I have any concerns. I have finished in my side.Reviewer #3: All concerns have been duly addressed. Information and narrative provided is sufficient. The data provided supports the conclusion provided.**********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  digital diagnostic tool,  14 Dec 2020AUTHORS’ RESPONSE TO EDITOR’S COMMENTSTitle: What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey dataDear Editor,Thank you for your email dated 10 December 2020 enclosing the Editor’s comments. We convey our gratitude to you for the comment that has led to the massive improvement of our paper entitled “What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey data”. We have now proofread the paper to correct grammar and spelling errors. All the changes are in tack changes in the revised manuscript. We believe the manuscript has improved substantively and will be published in your reputable journal. Version 2: PONE-D-20-23864R2Date: 11/12/2020Editor’s commentDear Dr. Budu,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Carefully proof your manuscript to correct grammar or spelling errors.Response: We have now proofread the paper to correct grammar and spelling errors Thank youSincerelyEugene BuduAttachmentResponse to Editors comments.docxSubmitted filename: Click here for additional data file. 17 Dec 2020What influences home delivery among women who live in urban areas in Ghana? Analysis of 2014 Ghana Demographic and Health Survey dataPONE-D-20-23864R2Dear Dr. Budu,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Frank T. SpradleyAcademic EditorPLOS ONE 23 Dec 2020PONE-D-20-23864R2 What influences home delivery among women who live in urban areas? Analysis of 2014 Ghana Demographic and Health Survey data Dear Dr. Budu:I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time.  Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus. Staphylococcus aureus is a Gram-positive, opportunistic pathogen which colonizes 30% to 50% of the human population  was a leading cause of health care-associated infections in the United States, with 323,700 estimated cases in hospitalized patients and 10,600 MRSA-related deaths  , which recognizes and binds the PG (Peptidoglycan hydrolases (PGHs) may represent such an alternative antibacterial agent . These e. aureus . It conss the PG . Endolyss the PG . Their ms the PG .d-alanine-glycine peptide bond, followed by a centrally located amidase domain, which cleaves the N-acetylmuramoyl-l-alanine amide bond within the PG , cytosolic (intracellular buffer), and lysosomal  conditions. This initial screening yielded a selection of 36 PGH constructs (approximately 11% of the entire library) that exceeded a relative activity score of 0.6, displaying robust staphylolytic activity throughout the tested conditions ; SEP, phage SEP1 endolysin; f11, phage phi11 endolysin; GH15, phage GH15 endolysin; LysH5, phage H5 endolysin; ALE1, bacteriocin ALE1; LST, lysostaphin; Xa, factor Xa protease cleavage site; TEV, tobacco etch virus cleavage site; PTD, protein transduction domain; H, His tag. Download FIG S1, PDF file, 0.2 MB.Relative activity scores of the 36 most potent staphylolytic PGHs identified by microtiter plate-based screening simulating extra- and intracellular conditions. Composite scores are the sum of individual scores derived from activities in 4 different buffers or media. Constructs are named according to their domain structure and origin of individual domains. CHAP, CHAP endopeptidase domain; M23, M23 endopeptidase domain; Pep, endolysin-derived endopeptidase domain; Ami, Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro activity assays, namely, a time kill assay (TKA), turbidity reduction assay (TRA), and spot-on-lawn assay (SLA)  (S. aureus.We expressed and purified the 36 PGHs identified by our screening approach and quantitatively compared them in three different reening) , which mreening) , a propeEscherichia coli, and purified. At this stage, His tag-free versions of all constructs were used to avoid possible biases. All proteins containing the CPP KalaSyn, as well as LysK, KLST, and CKAK fused to certain CPPs such as Pvec, were found to be either insoluble or insufficiently expressed and were therefore excluded from further analysis. The in vitro antibacterial activities of the remaining PGH-CPP fusion proteins were compared with those of the respective parental enzymes  were fused to various CPPs , cloned, enzymes . In gene enzymes . Therefo3+). When A549 epithelial cells were incubated with different concentrations of europium-labeled PGHs for 4 h, we observed a dose-dependent uptake of all proteins. Notably, a higher uptake rate was observed for the TAT-fused versions of all three proteins than that for the parental PGHs amino]biphenyl-4-yl}-2,2′:6′,2″-terpyridine-6,6′′-diyl}bis-(methylenenitrilo)}tetrakis(acetato)} europium(III)  infected with three different S. aureus strains . We screened for killing of S. aureus in this broad selection of cell lines to find PGH-CPP constructs with the ability to kill intracellular S. aureus regardless of the host cell tissue type. The selected cell lines originate from different tissues/organs within the body, which can all be infected by S. aureus, namely, lung, bone, and connective tissue.To test whether the increased cellular uptake of CPP-fused PGHs translates into enhanced killing of intracellular S. aureus cells were inactivated by the addition of flucloxacillin before treatment with the PGHs. When infected cells were exposed to LST and its CPP-containing derivatives for 4 h, we consistently found the intracellular killing activity of the CPP-fused variants to be significantly higher than that of the parental enzyme (S. aureus (numbers below the detection limit) was achieved with CPP-fused LST variants in the majority of the cell line-strain combinations -expressing S. aureus strain were exposed to LST, LST_TAT, or PBS and monitored by confocal laser scanning microscopy (CLSM). In these experiments, both LST and LST-TAT caused a reduction in the number of intracellular S. aureus bacteria compared to the control . This points toward a rapid release of cytosolic GFP upon lysis of S. aureus and is in agreement with the active killing mechanism of PGHs.To visualize the lysis of intracellular  control  to S3. A10.1128/mBio.00209-20.8MOVIE S1S. aureus and treated with PBS (control treatment). The GFP-expressing S. aureus RN9623 (green) was used for infection of MG-63 cells, and flucloxacillin was added to the medium after infection. DNA was stained with Hoechst 33342 (blue) and cell membranes with FM464 (red). Treatment with PBS was initiated at t = 0 h. To demonstrate intracellular localization of S. aureus, the red channel was imaged at t = 0 h but not used afterwards to minimize additional photobleaching. z-stacks were recorded every 5 minutes for 5 h. Scale bar = 30 μm. Download Movie S1, AVI file, 8.4 MB.Time-resolved three-dimensional CLSM video of eukaryotic cells infected with Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus in the coculture models. The effects were dependent on the eukaryotic cell line used, with the most significant reductions in intracellular CFU observed in the 3T3-L1 adipocytes  and their TAT-containing derivatives. For all three PGHs, increased killing in MG-63 cells was observed after 15 h, with GH15-TAT, SEP, SEP-TAT, and LysK significantly reducing intracellular  control . It is wS. aureus in our coculture model. Subsequently, we compared the efficacies of each tested pair of individual enzymes at these predetermined concentrations to that of a mixture of both enzymes at half the concentrations. In all cases, the PGH mixture killed significantly more bacteria than did the single PGHs , 20 μg (pink squares), and 100 μg (dark-pink squares), and cocktail_2_TAT was tested at 4 μg (light-blue squares), 20 μg (blue squares), and 100 μg (dark-blue squares). All animals received 1 mg flucloxacillin (F) at days 2 and 3 postinfection. (a to c) Total S. aureus numbers in the abscesses after 4 days (a) were determined as the sum of bacterial numbers in pus (b) and the surrounding tissue (c). (d) Relative numbers of intracellular bacteria in pus after treatment of pus with flucloxacillin ex vivo. (e) Relative mean bodyweight of mice treated with cocktail_1_TAT (pink squares), cocktail_2_TAT (blue squares), or buffer (black circles) over the time of infection and treatment. Error bars show the standard error of the mean (SEM). The administration of PGHs and flucloxacillin is indicated by black and gray arrows, respectively. Download FIG S2, PDF file, 0.4 MB.Efficacy of different doses of cocktail_1_TAT and cocktail_2_TAT against Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 8 CFU (inoculum) to 1.3 × 109 CFU during the course of the experiment indicates that S. aureus Cowan was able to proliferate in the mice despite flucloxacillin treatment. The synergism of LST_TAT and SEP_TAT that we had demonstrated in the cell culture model was also apparent in vivo, since the treatment with the individual PGHs was significantly less effective than with cocktail_2_TAT  S. aureus by PGHs.We therefore chose cocktail_2_TAT to further analyze its therapeutic potential in the murine abscess model, in comparison with its individual components (LST_TAT and SEP_TAT), as well as the respective parental  PGHs LST and SEP, alone and in combination (cocktail_2). As shown in il_2_TAT  and c. I the pus . This fi the pus  and d. I32.7 mm3 . As prev32.7 mm3 . Taken t10.1128/mBio.00209-20.3FIG S3S. aureus numbers in pus and tissue of murine abscesses. Animals were treated with 100 μg of LST (green squares), LST_TAT (dark-green triangles), SEP (orange inverted triangles), SEP_TAT (dark-orange diamonds), cocktail_2 , and cocktail_2_TAT (navy-blue squares) on days 1, 2, and 3 after infection. All animals including control mice (black circles) received 1 mg flucloxacillin (F) at days 2 and 3 after infection. (a and b) Numbers of viable S. aureus after the different treatments were determined in the pus (a) and tissue (b). The pound symbol (#) indicates a statistically significant difference between cocktail_2_TAT and all other treatments (P ≤ 0.0001 for panel a and P ≤ 0.05 for panel b). At least seven representative CLSM images of each of the four independent pus smears per treatment group were evaluated for total S. aureus numbers, eukaryotic cell numbers, and intracellular S. aureus. (c and d) Analyses are shown for total bacteria per square millimeter and intracellular bacteria per host cell. Asterisks (*) indicate levels of significance . Download FIG S3, PDF file, 0.3 MB.Analysis of Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. aureus. The different, highly complex environments found inside and outside cellular compartments pose diverse challenges to PGH activity, as they vary in multiple parameters such as pH, ionic strength, and osmolality  was more effective in this model than was cocktail_1_TAT, which instead contained GH15, an enzyme much less active in acidic environments   , effection (MBC) . Anothereptidase , 49.in vivo , but that the non-LST components within these mixtures  have an important impact on the potency of the cocktails. This is further supported by the relatively weak effects of LST and LST_TAT when applied individually in vivo , an obsein vivo . One reain vivo . Our celreatment . This efreatment .S. aureus has the striking ability to acquire new resistances and develop strategies to survive under stress conditions. The importance of intracellular S. aureus for recurrent infections is evident  and/or in abscesses  at 37°C. For the GFP-expressing S. aureus RN9623 (E. coli strains used for protein expression were grown at 37°C in Luria-Bertani (LB) medium  and on LB agar (LB medium plus 14 g/liter agar) supplemented with suitable antibiotics where necessary .All bacterial strains used in this study are indicated in s RN9623 , media w10.1128/mBio.00209-20.5TABLE S2Table S2, PDF file, 0.2 MB.Bacterial strains used in the present study. Download Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli BL21-Gold(DE3), and their construct identity was confirmed by commercial Sanger sequencing . A list of the plasmids used and created in this study is presented in The plasmid constructs used in this work were generated using standard molecular cloning techniques . DNA fra10.1128/mBio.00209-20.6TABLE S3Table S3, PDF file, 0.2 MB.PCR primers used in the present study. Download Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00209-20.7TABLE S4Table S4, PDF file, 0.2 MB.Plasmids used and created in the present study. Download Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains carrying the constructs of interest were performed in 96-well plates, as previously described  without calcium and magnesium , Dulbecco’s modified Eagle’s medium , intracellular buffer   supplemented with 1% albumin fraction V . Lysosomal buffer was produced by adjusting the pH of extracellular buffer . A score between 0 and 3 was assigned for each spot, and the scores at each of the three positions on the plate and of each buffer/medium were summed up to obtain a microtiter plate score for each PGH, as follows:m is the medium or buffer, p is the position, and PGHScore is {0, 1, 2, 3}, with 3 for complete clearance (0 CFU), 2 for 1 to 15 CFU, 1 for >15 CFU with visible single colonies, and 0 for >15 CFU without visible single colonies or bacterial lawns.We screened 322 PGH constructs from our library using a previously described microtiter plate-based approach  in an efescribed . Each PGith KOH)  supplemer buffer  to 4.7 uThe 36 PGHs with the highest activity across all four buffers/media were selected for further characterization.E. coli and purified essentially as previously described (600) of 0.5 at 37°C under agitation in LB medium modified for protein expression   and continued for 18 h at 19°C under agitation. Cells were harvested by centrifugation, and pellets were frozen at −80°C.Recombinant proteins were expressed in escribed . In briepH 7.8])  and coolE. coli was resuspended in lysis buffer  and lysed in a Stansted pressure cell homogenizer  at 100 MPa. The lysate was cleared of cellular debris by centrifugation and incubated with low-density nickel resin  in a gravity flow column. After washing, proteins were eluted with elution buffer , and fractions containing protein were identified using a NanoDrop ND-1000 spectrophotometer . Pooled fractions were dialyzed in Spectra/Por 1 dialysis tubing, with a 6- to 8-kDa molecular weight cutoff  against two changes of dialysis buffer  and consequently sterile filtered (0.2-μm pore).For constructs containing His tags, 2PO4, 20% glycerol [pH 7.4]) was used instead of lysis buffer. After clearance from the cellular debris, the lysate was sterile filtered and loaded onto a HiTrap Sepharose (SP) fast-flow (FF) cation exchange column  in an Äkta fast-performance liquid chromatography (FPLC) device . After washing with 3 column volumes of buffer A, a gradient of 1%/min of buffer B  was used to elute the protein. Fractions of 0.5 ml were collected, and those containing protein were dialyzed as described above.For constructs without a His tag, buffer A . All purified proteins were stored on ice at 4°C.To test for protein identity and purity, 4 μg of protein was analyzed by SDS-PAGE using Mini-Protean TGX-stain-free precast gels . Impure preparations underwent size exclusion chromatography in a Superdex 200 10/300 GL column  using gel filtration buffer  at this time point were scored with 4, 3, 2, and 1, respectively. The scores obtained for both tested enzyme concentrations were added and further processed as described above.TKAs were performed essentially as previously described . The fouS. aureus Newman substrate cells in each buffer, and 100 μl of the bacterial suspensions was mixed with 100 μl of each dilution of the PGHs in a 96-well plate, so that the initial OD600 of the suspension was 1. The decrease in optical density over time was monitored for 1 h at 30-s intervals with a FLUOstar Omega plate reader . The resulting lysis curves were corrected for the negative controls (no enzyme) and fitted to a 5-parametric sigmoidal function, as described before  with 10% fetal bovine serum  (l-glutamine) with 10% FBS (2 and maintained at ≤90% confluence for A549 and MG-63 cells and ≤80% confluence for 3T3-L1 cells. Differentiation of 3T3-L1 cells was performed as described before (A549 (ATCC CCL-185) adenocarcinomic human alveolar basal epithelial cells (A549 cells) and 3T3-L1 ATCC CL-173) primary murine fibroblast-derived adipocytes (3T3-L1 cells) were grown in Dulbecco’s modified Eagle medium  , 73. MG- primary  10% FBS . Cells wd before , with th3+) chelate was performed essentially as described before  in 0.1 M sodium acetate buffer (pH 4.9) were mixed with five parts of 17.2 mg/ml cyanuric chloride in acetone for 30 min at room temperature. The mixture was precipitated in pure acetone in a dropwise manner and washed with acetone two times, followed by vacuum drying for 1 h. Synthesis of the DTBTA-Eu3+ molecule was confirmed by electrospray ionization-mass spectrometry (ESI-MS) at the Functional Genomics Center Zurich . ESI-MS analysis was performed within a mass range between 50 and 5,000 Da, with a sampling cone energy of 40 V. Proteins were dialyzed into carbonate buffer (300 mM NaCl [pH 9.1]), and DTBTA-Eu3+ was mixed with purified proteins at a molar ratio of 4:1 and incubated for 2.5 h under agitation at room temperature. Unbound label was removed by gel filtration chromatography on a Sephadex G-25 gravity flow column . Protein concentrations were determined using the Pierce bicinchoninic acid (BCA) protein assay kit , according to the manufacturer’s instructions, and protein purity was confirmed by SDS-PAGE. Labeled proteins were incubated with A549 cells grown in a 24-well tissue culture plate  at concentrations of 0.5, 1.0, and 2.0 μM for 4 h. Cells were then washed three times with medium (DMEM or MEM) to remove PGHs. Cell integrity was confirmed by microscopy before and after washing. Eukaryotic cells were detached by adding 60 μl trypsin  and lysed by adding 240 μl of 0.1% Triton X-100  for 3 min at 37°C. Additionally, the mixture was mashed by repeatedly pipetting up and down. A 2-fold dilution series of each labeled PGH was prepared in the same buffer. Time-resolved fluorescence of the lysate and the dilution series (for generating a standard curve) was measured in a 384-well plate in an Infinite M1000 reader . PGH concentrations in the lysate were calculated from the standard curve.Labeling of PGHs and PGH-CPP fusion proteins with the fluorescent {{2,2′,2″,2‴-{4′-{[amino]biphenyl-4-yl}-2,2′:6′,2″-terpyridine-6,6′′-diyl}bis-(methylenenitrilo)}tetrakis(acetato)} europium(III)  of 1 for 1 h. At the start of infection, 225 nM death-associated protein kinase (DAPK) inhibitor  and 20 μM (3S)-5--3-[[(2S)-3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid (Q-VD-OPh)  were added to minimize the cytotoxic effects of S. aureus. Subsequently, eukaryotic cells were washed with prewarmed medium, and medium supplemented with 1 mg/ml flucloxacillin was added for 1 h to kill all extracellular bacteria  equipped with an HCX PL Fluotar 63× 1.30 oil objective. A z-stack of 20 μm with a z-slice thickness of 0.5 μm was imaged every 5 min, thereby creating time-resolved 3-dimensional reconstructions of the infected cells over a treatment duration of >4 h. Hoechst 33342, GFP, and FM4-64 were excited at λ of 405 nm, 488 nm, and 532 nm, respectively. Image analysis was performed with the Imaris Software .In each well of a 2-well imaging chamber , 1.25 × 10bacteria , 59. The5 A549 or MG-63 cells were seeded 24 h before infection. For the 3T3-L1 cell line, 6.25 × 104 cells were seeded in a 24-well plate 7 days before infection and differentiated as described above. On the day of infection, a log-phase culture of S. aureus Newman, Cowan, or USA300 JE2 was washed once and resuspended in PBS. The MOIs used for the infection of eukaryotic cells were 10, 1, and 1, respectively. The higher MOI for Newman was chosen because of its low propensity to invade eukaryotic cells, which is most likely due to the truncation of fibronectin binding proteins , and incubated with medium supplemented with 1 mg/ml flucloxacillin, which was determined to be at least 40× higher than the MIC of each strain. After 1 h, PGHs or PGH-CPP fusion proteins were added to the cells at 2 μM and incubated for 4 or 15 h at 37°C and 5% CO2. Controls without S. aureus infection and without PGH treatment were included in the assay. Additionally, the extracellular medium was plated to confirm the absence of extracellular S. aureus. Cells were washed three times with medium (DMEM or MEM) to remove flucloxacillin and enzymes. Cell integrity was confirmed by microscopy, and eukaryotic cells were detached by adding 60 μl trypsin  and lysed by adding 240 μl of 0.1% Triton X-100  for 3 min at 37°C. Additionally, the mixture was mashed by repeated pipetting, and 700 μl of cold PBS was subsequently added to the mixture. The cell lysate was serially diluted in PBS, plated on tryptic soy agar (TSA) plates, and incubated at 37°C overnight. On the following day, emerging colonies were enumerated.In each well of a 24-well tissue culture plate , 2.5 × 10proteins . Cells wS. aureus USA300 JE2 as described above, with the following modifications. In a preliminary dose-response experiment, 2-fold serial dilutions ranging from 0.04 to 0.0025 μM for LST_TAT and from 4 to 0.25 μM for CHAPGH15_SH3bALE1_TAT, LysK_TAT, and CHAPSEP_SH3b2638a_TAT were added to the eukaryotic cells for 15 h. For all enzymes, concentrations yielding approximately the same log reduction in intracellular S. aureus numbers were determined. To test for synergy between LST_TAT and any of the other three PGHs, log reductions caused by two individual enzymes in the intracellular killing assay at these predetermined concentrations were compared with those caused by a mixture of the two proteins at half these concentrations.To determine the synergistic effects of mixtures of PGH-CPP fusion proteins inside eukaryotic cells, intracellular killing assays were performed in MG-63 cells infected with S. aureus USA300 JE2. In both cases, cells were treated with 2 μM PGHs for 15 h, as described for the intracellular killing assay. The absorption of the supernatant at 490 nm  and 680 nm (background) was measured with a FLUOstar Omega plate reader . After background subtraction, cytotoxicity was determined by division of the signal of the samples by the signal of a maximum LDH activity obtained through lysis of all cells by a detergent and subsequent multiplication by 100%.Cytotoxicity assays were performed according to the manufacturer’s instructions of the Pierce LDH cytotoxicity assay kit . Briefly, the supernatant of A549 cells was tested for its LDH content either with or without infection by The Institutional Animal Care and Use Committee of the University of Zurich approved the study under protocol ZH050/18, and all animal experiments conducted in this study were approved by the Cantonal Veterinary Office Zurich.S. aureus Cowan was grown to logarithmic phase, and 108 CFU were mixed 1:1 with Cytodex beads  and injected subcutaneously into the flanks of 7- to 8-week-old female C57BL/6 mice , as previously described  and with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody Ab150113 (Abcam). The samples were visualized as described above.In addition, abscess pus was stained with 10 μg/ml FM 4-64FX  and 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific) before fixation with 4% paraformaldehyde . PBS with 0.1% albumin fraction V , 2% FBS (Thermo Fisher Scientific), and 1% saponin (Sigma-Aldrich) was used to block and permeabilize the samples. in vivo experiments, the weight loss data of mice were analyzed by a two-way ANOVA with Dunnett’s correction for multiple comparisons. Differences in CFU counts and abscess size were analyzed using a one-way ANOVA with Tukey’s correction for multiple comparisons.All experiments were performed at least in biological triplicates, and statistical analysis was performed in Prism v8.0 . The CFU counts of all experiments were log transformed prior to analysis. A two-way analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons was used to analyze the difference in (i) the uptake of europium-labeled PGHs with or without CPPs, (ii) intracellular CFU after exposure to different PGHs with and without CPPs, and (iii) the difference in intracellular CFU after exposure to these agents for different periods (4 h and 15 h). Differences in intracellular CFU in synergy experiments were analyzed using a one-way ANOVA with Tukey’s correction for multiple comparisons. For The data supporting the findings of this study are available within the article and its supplemental material files, or from the corresponding author on request.10.1128/mBio.00209-20.9MOVIE S2S. aureus and treated with LST. The GFP-expressing S. aureus RN9623 (green) was used for infection of MG-63 cells, and flucloxacillin was added to the medium after infection. DNA was stained with Hoechst 33342 (blue) and cell membranes with FM464 (red). Treatment with LST was initiated at t = 0 h. To demonstrate intracellular localization of S. aureus, the red channel was imaged at t = 0 h but not used afterwards to minimize additional photobleaching. z-stacks were recorded every 5 minutes for 5 h. Scale bar = 30 μm. Download Movie S2, AVI file, 8.2 MB.Time-resolved three-dimensional CLSM video of eukaryotic cells infected with Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00209-20.10MOVIE S3S. aureus and treated with LST_TAT. The GFP-expressing S. aureus RN9623 (green) was used for infection of MG-63 cells, and flucloxacillin was added to the medium after infection. DNA was stained with Hoechst 33342 (blue) and cell membranes with FM464 (red). Treatment with LST_TAT was initiated at t = 0 h. To demonstrate intracellular localization of S. aureus, the red channel was imaged at t = 0 h but not used afterwards to minimize additional photobleaching. z-stacks were recorded every 5 minutes for 5 h. Scale bar = 30 μm. Download Movie S3, AVI file, 8.2 MB.Time-resolved three-dimensional CLSM video of eukaryotic cells infected with Copyright © 2020 Röhrig et al.2020Röhrig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the