Routine human papillomavirus (HPV) testing is performed in cervival cancer and is required for classification of some head and neck cancers. In penile cancer a statement on HPV association of the carcinoma is required. In most cases p16 immunohistochemistry as a surrogate marker is applied in this setting. Since differing clinical outcomes for HPV positive and HPV negative tumors are described we await HPV testing to be requested more frequently by clinicians, also in the context of HPV vaccination, where other HPV subtypes are expected to emerge.Therefore, a cohort of archived, formalin-fixed paraffin embedded (FFPE) penile neoplasias was stained for p16 and thereafter tested for HPV infection status via PCR based methods. Additionally to Sanger sequencing, we chose LCD-Array technique  for the detection of HPV in our probes expecting a less time consuming and sensitive HPV test for our probes.We found that LCD-Array is a sensitive and feasible method for HPV testing in routine diagnostics applicable to FFPE material in our cohort. Our cohort of penile carcinomas and carcinomas in situ was associated with HPV infection in 61% of cases. We detected no significant association between HPV infection status and histomorphological tumor characteristics as well as overall survival.We showed usability of molecular HPV testing on a cohort of archived penile carcinomas. To the best of our knowledge, this is the first study investigating LCD-Array technique on a cohort of penile neoplasias.The online version contains supplementary material available at 10.1186/s12894-022-00962-4. Infection by human papillomavirus (HPV) is associated with a variety of cancers including anal squamous cell carcinomas (SCC), oropharyngeal cancers, cervical cancers, vulvar and vaginal cancers as well as penile carcinomas . Penile We used p16 immunohistochemistry, Sanger sequencing and the LCD-Array hybridisation technique (Chipron) for HPV detection on a cohort of penile squamous cell carcinomas, carcinomas in situ and peritumoral tissue and compared the sensitivity and specificity, respectively. We chose LCD-Array hybridisation technique (Chipron) in direct comparison to Sanger sequencing expecting reduced hands-on time and easy interpretation of the results. To shed light on the question of which test is the most efficient one for daily routine, we compared the above mentioned three distinct tests. To our knowledge, this is the first study testing LCD array technique on a cohort of FFPE material of penile neoplasias.Between 2002 and 2017 70 cases with sufficient FFPE material were identified with penile squamous cell carcinomas, squamous cell carcinoma in situ (Cais) of the penis, extramammary Paget disease, focally invasive as well as basal cell carcinoma of the penis. The paraffin blocks were provided by the Dr. Senckenberg Institute of Pathology, University Hospital, Frankfurt, Germany.We systematically compared tumor and individual matching non-tumor probes for 69 of 70 cases. In a single case, a lymph node metastasis was investigated. Therefore, we applied a total of 139 tests, for one probe only metastatic material was available. In total, we investigated 62 invasive squamous cell carcinomas, 6 squamous cell carcinomas in situ, one basal cell carcinoma, and one extramammary Paget disease, focally invasive. Additionally, peritumoral epithelium was tested, wherever available. The peritumoral epithelium was macrodissected or taken from another block wherever available.Median age at diagnosis was 65 years (range 41–85 years). The lesions were mostly located at the glans penis (n = 50), followed by the foreskin (n = 9) and shaft (n = 2). In 9 cases, the primary tumor localisation could not be determined due to either a large tumor size or incomplete documentation.Penile carcinomas were staged and characterized according to TNM staging and the 8th edition of the classification system of the Union for International Cancer Control (UICC) . The tumTissue samples and patient data used in this study were provided by the University Cancer Center Frankfurt (UCT). Written informed consent was obtained from all patients and the study was approved by the institutional review board of the UCT and the ethical committee at the University Hospital Frankfurt (project-number: SUG-02-2017) according to the declaration of Helsinki. For our studies, archived material was used in a double pseudonymised manner. Diagnostics were already finalised by the time of study.All cases were stained with an antibody against p16. Briefly, freshly cut 1 µm thick paraffin sections were stained using CINtec®  according to manufacturers’ instructions. Immunohistochemistry was performed using the DAKO FLEX-Envision Kit  and the fully automated DAKO Omnis staining system  according to manufacturer´s instructions. Epitope retrieval was generated at pH6 and 97 °C. Epitope staining was applied for 30 min. Epitope visualization was done by DAKO EnVision™ FLEX DAB + Substrate Chromogen System . DAKO haematoxylin solution  was used for nuclear counterstaining. Positive staining was noted in the case of block like positive staining. Mosaic staining pattern was noted as negative.Tumor and non-tumor samples were macrodissected from 10 µm thick paraffin sections that were freshly cut. DNA extraction was performed applying the Maxwell 16 FFPE tissue LEV DNA purification kit . Quantification was performed using Quantus Fluorometer . DNA quality was assessed by fragment analysis . HPV subtypes were analysed by Sanger sequencing and compared to LCD-Array technique .Nested polymerase chain reaction (PCR) using GP5+/6+ and MY09/11 primers, commonly used for HPV detection, were tested on a subset of probes as described previously . Primer https://www.chipron.com). PCR settings were the following: Start at 96 °C for 3 min, 42 times 94 °C for 1 min, 45 °C for 1 min and 30 s, 72 °C for 1 min and 30 s, finalized by 72 °C for 3 min and termination at 4 °C. The following HPV subtypes can be detected: 6, 11, 16, 18, 31, 33, 35, 39, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 70, 72, 73, 81, 82, 83, 84, 90 and 91. Tumor and non-tumor samples were tested in pairs. One positive and one negative control were taken per run. Figure All samples were re-analysed by applying HPV 3.5 LCD-Array Kit , according to manufacturers’ instructions  was investigated. Two samples inherited either metastasis or central biopsy probe. All tumor and non-tumor specimens were screened, wherever applicable, for p16 and HPV status .Most of the invasive squamous cell carcinomas were stage pT1a and pT2. Six cases displayed Carcinoma in situ  and two cases  consisted of extramammary Paget disease, focally invasive and basal cell carcinoma.In our cohort we detected exceedingly more squamous cell carcinomas, usual type , then squamous cell carcinomas, basaloid type 6/60, 10%). In many cases lymph nodes were not removed . Most cases displayed no (lympho-)vascular invasion  and no perineural invasion . In most cases tumor grading was determined as G2  on a scale G1–G3 . In total block like p16 positivity was detected in 39/138 (28%) evaluable samples.Invasive SCCs (n = 62) were p16 positive in 32 cases . Carcinoma in situ were positive in 4/6 cases (67%). Extramammary Paget disease, focally invasive and basal cell carcinoma resulted negative for p16 staining.For non-neoplastic epithelium 68  cases were evaluable. Peritumoral epithelium was positive for p16 immunohistochemistry in 3 cases . All stainings are displayed in Additional file Overall, 137 samples consisting of tumor and non-tumor probes could be evaluated  by Sanger sequencing. HPV infection was detected in 21 samples .61 of 62 (98%) invasive squamous cell carcinomas were evaluable via Sanger sequencing. 16/61 (26%) invasive carcinomas were positive for HPV infection: 13 cases showed positivity for HPV 16 . The remaining 3 cases harboured 2 co-infections HPV 16 and 33  and one HPV 33 infection .For carcinoma in situ (n = 6) all cases were evaluable . 1 case showed HPV infection  with HPV 16. The remaining 5 cases were negative for HPV infection .Extramammary Paget disease, focally invasive and basal cell carcinoma were negative for HPV infection by Sanger sequencing.Matching non-neoplastic epithelium was evaluable in 68/69 (99%) cases. 4/68 (6%) cases harboured HPV infection by HPV 16.In total 134/139 (96%) samples showed sufficient material for LCD-Array analysis. Of these HPV infection was detected in 58 probes .With LCD-Array technique, 60/62 (97%) invasive carcinomas were evaluable. 34/60 (57%) probes tested were positive for HPV infection. Among these, HPV 16 was the most frequent: 26/60 (43%). Eight remaining cases harboured infections by HPV 11 , HPV 31 , HPV 33  and 4 cases showed co-infections of HPV 16 and 44, HPV 16 and 58, HPV 16 and 70 as well as HPV 18 and 53 . The results are summarised in Fig. Carcinoma in situ (n = 6) were tested positive for HPV infection in 100% of cases (6/6). 5/6 (83%) showed HPV 16 mono-infection. One case showed co-infection with HPV 16 and 33 .Extramammary Paget disease, focally invasive and Basal cell carcinoma were both negative for HPV infection via LCD-Array technique.For non-neoplastic epithelium, 66/69 (96%) were evaluable. 18/66 (27%) showed infection by HPV: HPV 16 was proven in 16/66 (24%) of cases. The remaining two cases harboured an infection by HPV 11 and a co-infection of HPV 16 and 33 .Results for p16, Sanger sequencing and LCD-Array technique are summarised in Table https://www.aid-diagnostika.com/) by strip hybridization (AID Diagnostica) with 100% concordance.Five cases with positive results in peritumoral epithelium via LCD-Array technique were additionally investigated at another lab . Overlap means that a positive p16 staining results in a HPV subtype determination via Sanger sequencing and LCD-Array and vice versa: negative p16 staining should lead to negative HPV proof by the other techniques.The overlap between p16 and Sanger sequencing was 103/129 (80%) cases, the overlap between p16 and LCD-Array technique was 108/129 (84%) cases, and between Sanger sequencing and LCD-Array technique was 92/129 (71%) cases.LCD-Array technique taken as gold standard lead to a sensitivity of 63% and a specificity of 100% for p16 staining, respectively. Prevalence was 43%. The rate of false positives was 0%, the rate of false negatives was 38%. Positive predictive value was 100%, negative predictive value was 78%.Taking LCD-Array technique as gold standard Sanger sequencing had a sensitivity of 34% and a specificity of 100%. Prevalence was 43%. The rate of false positives was 0%, the rate of false negatives was 66%. Positive predictive value was 100% and the negative predictive value was 66%.For squamous cell carcinomas n = 57 samples contained measurements for all three techniques. The total overlap between all three techniques in squamous cell carcinomas was 39 .Concordant and discrepant results in squamous cell carcinomas are displayed in Fig. Looking at squamous cell carcinomas per se and taking LCD-Array as the gold standard, the prevalence for p16 positivity was 56%, sensitivity was 91%, specificity was 100%. The rate of false positives was 0%, the rate of false negatives was 9%. The positive predictive value of p16 staining was 100% and the negative predictive value was 89%.Comparing Sanger sequencing and LCD-Array technique, taking LCD-Array technique as the gold standard, prevalence was 56%, sensitivity was 47%, specificity was 100%. The rate of false positives was 0% and rate of false negatives was 53%. The positive predictive value was 100% and the negative predictive value was 60%.HPV status was related to tumor characteristics. Fisher’s exact test showed a tendency towards an association between HPV status and tumor stage for LCD-Array technique . The mean infiltration depth was 6 mm . There was no significant association between tumor size and infiltration depth in relation to HPV status obtained via LCD-Array technique  approved several HPV testing methods: APTIMA HPV 16 18/45 Genotype Assay and APTIMA HPV Assay , Cervista HPV 16/18 and Cervista HPV HR and Genfind DNA Extraction Kit , COBAS HPV Test and cobas HPV for use on the cobas 6800/8800 Systems , Digene Hybrid Capture 2 High-Risk HPV DNA Test  and BD ONCLARITY HPV ASSAY  [APTIMA HPV 16 18/45 Genotype Assay is indicated for the qualitative detection of E6/E7 viral messenger RNA (mRNA) of HPV types 16,18 and 45. It differentiates between HPV 16 and HPV 18 and/or HPV 45 . APTIMA A study comparing sensitivity and specificity of the above mentioned tests showed that all DNA/RNA-based tests, except the NorChip test, showed high sensitivity rates for high-grade lesions positive by cytology . These tThe LCD-Array’s performance was demonstrated by the HPV Laboratory Network (LabNet) in comparison to other tests with 100% proficient results . The advThe cohort of Frankfurt penile carcinomas and carcinomas in situ was associated with HPV infection in 61%  of the cases as defined by LCD-Array technique, which is in line with other studies , 15 and We were able to show HPV infection in peritumoral epithelium . Positive measurement in non-neoplastic epithelium resulted in positive measurement in neoplastic epithelium. The question needs to be raised, why, despite HPV infection in both fractions, one resulted in tumor growth and the other did not. The HIM Trial postulates up to 37 HPV types in the genitals, of which ca. 5% progressed to a genital lesion . It can As expected, the most frequent type of HPV infection in squamous cell carcinomas resulted from HPV 16. That is in line with previous studies .In the tumor cohort, we investigated squamous cell carcinomas of the usual type and basaloid subtype. Although the usual type is classified as non-HPV-related, according to the WHO classification, we found 30 samples with HPV infections among 54 evaluable tumor samples described as squamous cell carcinoma, usual type (56% positive samples), which is in line with a previous review . We haveNext generation sequencing (NGS) methods have been applied to tumor tissue and revealed subtypes not known before the advent of these techniques . AdditioHPV was also detected in lymph node metastases. There are approaches to detect circulating cell free HPV DNA as a measure for disease control in oropharyngeal cancer . This teIn the setting of the LCD-Array test 32 HPV types can be detected, which exceeds the types tested by the aforementioned tests. Currently it is not feasible to apply NGS methods to the large amount of samples observed in daily routine, as the costs exceed the possible compensation. This is why LCD-Array technique is a fast and reliable alternative for routine practice.p16 staining is in most cases sufficient for the detection of high risk HPV association of penile neoplasias. LCD-Array is a feasible, sensitive and specific, as well as cost efficient diagnostic tool for HPV testing of FFPE tissue of penile cancer which can be applied in routine testing in addition to p16 staining or in clinical trials. LCD-Array technique may serve as a helpful tool in the advent of other HPV subtypes emerging in the setting of HPV vaccinated patients. Readouts of molecular techniques must be interpreted with caution to not over interpret transient HPV infections in the setting of a positive HPV detection in non-neoplastic epithelium.Additional file 1: Fig. 1. Negative p16 stainings.Additional file 2: Fig. 2. Positive p16 stainings.Additional file 3: Fig. 3.A Kaplan–Meier-Estimator for p16 positive and negative staining for overall survival in the cohort of penile neoplasias . B Kaplan–Meier-Estimator for LCD-Array with regard to HPV high risk positive and HPV high risk negative cases for overall survival in the cohort of penile neoplasias . C Kaplan–Meier-Estimator for Sanger sequencing with regard to HPV high risk positive and HPV high risk negative cases for overall survival in the cohort of penile neoplasias .Additional file 4: Table 1a. HPV status of invasive squamous cell carcinomas by p16 immunohistochemistry with regard to tumor characteristics in evaluable cases**. Table 1b. HPV status by Sanger sequencing technique with regard to tumor characteristics in evaluable cases**.


Papillomaviridae are not well understood. Using a combination of k-mer distributions, genetic metrics, and phylogenetic algorithms, we sought to evaluate the characteristics and differences of Alpha-, Beta- and Gamma-PVs constituting the majority of HPV genomes. A total of 640 PVs including 442 HPV types, 27 non-human primate PV types, and 171 non-primate animal PV types were evaluated. Our analyses revealed the highest genetic diversity amongst Gamma-PVs compared to the Alpha and Beta PVs, suggesting reduced selective pressures on Gamma-PVs. Using a sequence alignment-free trimer (k = 3) phylogeny algorithm, we reconstructed a phylogeny that grouped most HPV types into a monophyletic clade that was further split into three branches similar to alignment-based classifications. Interestingly, a subset of low-risk Alpha HPVs  split from other HPVs and were clustered with non-human primate PVs. Surprisingly, the trimer-constructed phylogeny grouped the Gamma-6 species types originally isolated from the cervicovaginal region with the main Alpha-HPV clade. These data indicate that characterization of papillomavirus heterogeneity via orthogonal approaches reveals novel insights into the biological understanding of HPV genomes.Papillomaviruses (PVs) are a heterogeneous group of DNA viruses that can infect fish, birds, reptiles, and mammals. PVs infecting humans (HPVs) phylogenetically cluster into five genera , with differences in tissue tropism and carcinogenicity. The evolutionary features associated with the divergence of  Papillomaviruses (PVs) are a heterogeneous set of double-stranded DNA viruses with a circular genome of approximately 8000 bp. PVs mainly infect epithelial surfaces, for example, anogenital skin and mucosa, of a large spectrum of host species including humans, monkeys, reptiles, birds, aquatic, and terrestrial vertebrates, and have co-evolved with their host species for millions of years ,2. PV inhttp://www.ictvonline.org/; accessed on 31 October 2018) [The PV genome has served as the basis for classification and understanding the biology and disease association of this ancient and diverse set of viruses. The designation of a PV type represents the fundamental taxonomic level of a highly related group of viruses that have similar genetic and biologic characteristics ,7,8. Curer 2018) . HPVs cler 2018) ,11,12. Ter 2018) . Howeverer 2018) ,20,21, ier 2018) ,23,24,25er 2018)  as well er 2018) ,28. Thuser 2018) . Characterizing potential mechanisms driving genetic diversity upon which selection and fixation occurs is important for understanding viral associated diseases and basic principles of genome evolution. Since PVs rely on host enzymes for viral gene expression and replication, virus-host biological interactions are critical for viral fitness. It has been reported, for example, that HPVs have a strong codon usage bias different from their human hosts ,31. Codok-mer: a DNA ‘word’ with fixed length) and additional genetic metrics  provide complementary information on the complexity and relationships of PV genomes [With advances in DNA sequencing and bioinformatics analyses, multiple sequence alignments have been used to interrogate PV genomes using parametric methods. For example, incongruent tree topologies observed between early and late gene phylogenies suggested potential recombination events in Alpha-, Omicron- and Upsilon-PVs implying that rare events may contribute to the complex evolutionary history in part obscuring simple host–parasite coevolution ,36,37,38 genomes ,40,41,42 genomes . In an effort to interrogate evolutionary features associated with the divergence of papillomaviruses generally, and HPVs specifically, we analyzed genomic features between Alpha-, Beta-, and Gamma-PVs constituting the majority of HPV genomes. We have primarily focused on PVs from a single host (human) to avoid strong host biases, and to try to observe genomic features possibly associated with phenotypic outcomes such as cancer. Multiple parametric and nonparametric algorithms were applied. Using the characterization of a large number of PV genomes, we sought to uncover hidden biological signals not easily discovered with homology-based methods alone.Firstpapillomavirinae within the family Papillomaviridae [p < 0.001) and Beta-PVs  (pi = 0.37 ± 0.03) and the multipurpose DNA helicase E1 (pi = 0.40 ± 0.04) were the most conserved genes compared to other genes (0.53 ± 0.06 > pi > 0.43 ± 0.04). Alpha-HPVs are constituted by three monophyletic branches including one high-risk clade  and two low-risk clades   . Distinc< 0.001) . The maj and 14) . k = 3) spectra. This analysis has the potential to reveal “higher order” features of viral genomes in comparison with the alignment-based phylogeny that relies primarily on protein coding regions [In order to explore an agnostic evolutionary model of HPV genomes, we constructed a phylogeny using trimer ( regions . As showp < 0.001)  separated from the main cluster of HPVs and sorted to the clade containing the majority of non-human primate PVs (in black) and non-primate animal PVs (in grey) within Group 2 . Althoug< 0.001) .p < 0.001). In contrast, Gamma-HPVs had the lowest ENC values (45.1 ± 2.0), followed by Alpha-HPV-HR (46.2 ± 2.0), Alpha-HPV-LR1 (48.7 ± 1.5) and Beta-HPV (48.7 ± 2.0), although a phylogenetic generalized linear model test (pglm) did not support statistical significance between HPV groups (p = 0.1621). Consistent with the trimer distribution clustering, macaque Alpha-PVs codon usage did not differ from the Alpha-HPV-LR2 types   values across 6 open reading frames (ORFs) to compare the codon usage bias between different HPV phylotypes  . Only of938) see .To pursue explanations for the trimer distribution analysis tree, we looked at the relationship between codon usage bias and ORF sequence composition. As shown in Besides the codon usage measurement of an entire genome and/or gene, we calculated the Relative Synonymous Codon Usage (RSCU) values of HPV genomes. This analysis estimates the differential usage of each synonymous codon. Among 59 codons encoding 18 amino acids , 6 codons were preferred  and 15 were suppressed  across the surveyed PV genomes . Most HP2 = 0.5773, pseudo F = 150.2, p < 0.001) was attributable to the differences between PV groups. Beta-/Gamma-HPV genomes predominantly infecting the skin regions clustered together , with a tendency to use more AGA encoding for Arginine when compared to the mucosal types (2 = 0.4295) and L1 (R2 = 0.3894) genes contained more variation in synonymous codon usage that results in the clustering of HPV taxa, followed by L2 (R2 = 0.3761), E2 (R2 = 0.2253), E6 (R2 = 0.2082) and E7 (R2 = 0.1120)  . HPV gen 0.1120) . In addip < 0.001) (p = 0.004) and TpA . We also observed substantial dinucleotide suppression between HPV genes suggesting that phenotypes and proteins sharing similar biological properties were often clustered  , consist< 0.001) . In contlustered . p < 0.001). The loss of CpG in Macaque-αPV and Alpha-HPV-LR2 genomes appeared to be positively associated with the gain of CpA (=TpG) and CpC (=GpG), respectively. The data suggest a potential role of the deamination of methylated C to T in CpG dinucleotide depletion, but mutational pressure, genetic drift and/or translational selection, could also be driving CpG suppression in HPV genomes.It has been reported that depletion of CpG dinucleotide could be the result of deamination of 5-methylcytosine (5mC) leading to a cytosine (C) to thymine (T) transition within a CpG island, resulting in a gain of CpA (=TpG) . MethylaIn this study, we applied multiple parametric and nonparametric algorithms including evolutionary phylogeny, trimer distributions, codon usage bias and dinucleotide suppression to systematically evaluate HPV genomic characteristics. Using the sequences of a large number of viral genomes, we provide evidence of differential evolutionary constraints associated with the heterogeneity of HPV genomes. Using an agnostic trimer analysis, we show that the Alpha-PVs are more disparate than assumed from homology-based analyses. In particular, the separation of a low-risk Alpha-HPV clade  from the main clustering of HPV genomes was consistently supported by trimer phylogeny, codon usage and dinucleotide suppression. These observations suggest that additional information in papillomavirus genomes, revealed by these analyses, could be under evolutionary pressures and contribute to HPV niche adaptation, host specificity, immune evasion, and pathogenicity ,55,56. Papillomaviradae. Our data indicated that k-mer distribution (k = 3), codon usage bias and dinucleotide suppression allow statistical analyses to identify sequence signatures of PV genomes corresponding to differences in host specificity (human vs macaque PVs), tissue tropism , and carcinogenic potential (high-risk vs low-risk HPVs). Viruses infecting a single host  have a tendency to contain similar genetic features. Within the genus Alphapapillomavirus, for example, we found two deeply separated groups  with significant differences in trimer distributions, codon usage and GC content. Strikingly, Gamma-6 (γ6) HPV types shared more “high order genomic features” with Alpha mucosal HPVs , Papillomavirus Episteme , GenBank/NCBI , and our own PV database when this analysis was initiated (31 October 2018). We found that there were 782 complete genomes assigned with unique virus names as well as NCBI accession numbers. A pairwise sequence comparison using the complete L1 ORF nucleotide sequences identified 653 genomes sharing less than 90% similarity with other PV genomes and representing distinct types. Eleven HPV metagenomic sequences lacking complete ORFs of E1, E2, L1 or L2 gene, ScPV1 (accession number of MF564196) with unusual E6/E7 ORFs, and SaPV1 (KX643372) classified into the subfamily Secondpapillomavirinae (mainly containing fish PVs) were excluded from further analysis in this study, resulting in a total of 640 PV complete genomes  composed of the frequencies of 64 3-bp forms across viral genomes were summarized to calculate a Kullback-Leibler (KL) distance matrix [count and hclust in R’s packages ‘seqinr’ [The term e k-mers . Given te matrix , based o‘seqinr’  and ‘sta‘seqinr’  were usehttp://codonw.sourceforge.net/; accessed on 31 October 2018) to calculate the ENC values that range between 20 (extremely strong bias as only a single codon for each amino acid is used) and 61 . We further plotted the relationship between ENC and GC3s (GC content at the synonymous third codon position) to examine the influence of GC content on codon usage. This was compared to the expected ENC* if GC content were solely responsible for the codon biases, calculated as [The effective number of codons (ENC) statistic is able to determine how biased a gene is in terms of its codon usage . We usedlated as ,48:ENC*=http://genomes.urv.cat/CAIcal/; accessed on 31 October 2018) was used to calculate the RSCU values. The RSCU values in the host genomes serve as the references, as retrieved from the Kazusa codon usage database   [The ENC is widely used to characterize codon usage bias. However, it does not handle each codon differentially. We calculated the Relative Synonymous Codon Usage (RSCU) values for 59 codons : the ratio of the observed frequency of codons relative to the expected frequency in the absence of usage bias to measure the extent of non-random usage of synonymous codons . If the CG represents the frequency of a dimer CG, fc and fG denote the probabilities of its constituent monomers, respectively. CpG suppression, the observation of lower-than-expected numbers of 5′-CG-3′ dinucleotides, is a common phenomenon in most portions of vertebrate genomes. This feature is likely to be due to C-to-T mutation, driven by CG-specific DNA methyl transferases. In viral genomes, suppression of CG dinucleotides may help them to evade host cellular defenses. We measured the dinucleotide suppression of the core dataset of 228 PV genomes including GpC and other 15 dimers, calculated as the observed frequency of the dinucleotide relative to the product of the frequencies of the individual nucleotides . The dinFor dinucleotides that do not form a reverse complemented pair on the opposite strand , for instance, the ratio is: rda function in R’s package ‘vegan’ was used to generate two-dimensional representations for matrix 1 and 2 and visualized using the biplot. Differences in codon usage bias were assessed with permutational multivariate analysis of variance (PERMANOVA), using adonis2 in R’s package ‘vegan’.The correlation between codon usage and PV genomic composition was tested using correspondence clustering approaches including multidimensional scaling of the redundancy analysis (RDA) and optionally principal coordinate analysis (PCoA) . The rdacor.test in R’s package ‘stats’). The significance of the differences in the genetic metric measures between HPV genes and genomes was tested using phylogenetic generalized linear models (pgls function in R’s package ‘caper’). All plotting and statistical comparisons were performed using R scripts developed in house [The agreement between distance matrices inferred from sequence alignment and trimer distribution of PV genomes was measured using the Mantel test. The Pearson’s correlation coefficient was calculated to test the association between dinucleotide suppression pairs (in house .


Among outgoing D1–D4 students, the one-hour HPV training increased participants’ self-perceived ability to describe the burden of HPV disease, discuss the importance of HPV vaccination for cancer prevention, and provide needed HPV vaccination information to parents . Conclusions: Understanding deficits in dental student knowledge and attitudes across the 4 years of dental school may help dental educators better understand the timing and content needed for effective HPV training in the dental school curriculum to reduce HPV-associated OPC prevalence.The purpose of the current study was to assess knowledge and attitudes about human papillomavirus (HPV) and HPV vaccination for oropharyngeal cancer (OPC) prevention in first-year through fourth-year dental students. Methods: In this cross-sectional quantitative study, surveys assessed knowledge and attitudes about HPV, HPV-related OPC, and HPV vaccination of incoming first-year dental students (D1), outgoing first- and second-year dental students (D1–D2), and outgoing third- and fourth-year dental students (D3–D4). After completing a 40-item pre-training knowledge and attitude assessment survey, a one-time, one-hour national HPV training session was conducted. An 8-item attitudinal post-survey was completed after training. Results: Of 173 participants (75.9% response rate), over 85% did not know that the rate of HPV is not highest among women in their 30s, and only 11% to 28% knew that smoking-associated OPC is more deadly than HPV-associated OPC. While participants overall expressed willingness to administer the HPV vaccine, the willingness of dental students to do so in their future practice tapered off progressively through dental school year categories ( Human papillomavirus (HPV) is the most prevalent sexually transmitted disease in America  and the The HPV vaccine has been effective at reducing the prevalence of HPV infection, including the high-risk HPV types 16 and 18 associated with oropharyngeal cancer (OPC) ,8. RecomThe ACIP recommendations allow for HPV vaccination as early as 9 years old . FurtherDespite the potential benefits of providing HPV vaccine counseling and recommendations in the dental office, studies of dentists ,5,14,15,The current study used a cross-sectional survey design, and all survey responses were anonymous. The study was conducted according to the guidelines of the Declaration of Helsinki, reviewed by the A.T. Still University Institutional Review Board and was deemed exempt. Electronic and paper-and-pencil surveys were completed by dental students before (pre-training) and after (post-training) a training session about HPV at two sites of a single US dental school. In March 2019, all outgoing first-year (D1), second-year (D2), third-year (D3), and fourth-year (D4) students were required to attend the one-time hour HPV training in collaboration with the Missouri Area Health Education Center HPV Immunization Project (a US Centers for Disease Control and Prevention-funded initiative). Each student was e-mailed a survey invitation that described the study and included a link to the electronic pre-training survey. The survey was compiled by the author JD and administered by the author CL. The first question of the 40-question survey addressed informed consent, and all students could choose to participate in the study or opt out. Students were advised that their participation was voluntary and that there were no negative consequences of non-participation. The one-hour training session provided instruction about HPV and the HPV vaccination. An onsite lunch was provided to encourage participation in the voluntary pre-training survey. After the one-hour training, dental students completed a voluntary 8-question post-training paper-and-pencil survey. In July 2019, incoming D1 students were invited to participate in the study by completing the electronic pre-training survey only. No training was provided at that time.The 40-question pre-training survey had three sections: demographics, 30 items assessing knowledge of HPV and OPC, and 10 items assessing attitudes about the scope and role of dental professionals in addressing HPV and OPC through counseling and administration of the HPV vaccine. Demographic items asked about sex, age, year in dental school, and race and ethnicity. Knowledge assessment included 18 items about HPV and OPC and 12 about HPV vaccination; response choices were  and one 5-level multiple choice. Attitude assessment asked about perceived responsibility, willingness, and understanding of HPV and OPC; these items used 4-point to 7-point Likert-like scales. An open text box was included to allow participants to provide comments or reflections about providing HPV vaccination information during a dental appointment. The students were given 30 min to complete the survey.The 7-item post-training survey used 5-point Likert scale items that estimated the effect of the training session directly, i.e., “as a result of this training”, and further, some items also included modifiers such as “better” and “more aware” (1 = strongly agree and 5 = strongly disagree). An additional question requested feedback or comments. The students had 30 min to complete the survey following the HPV training. Double-data entry was used to convert paper responses to digital format for analysis. The two copies were also compared to resolve any inconsistencies.Knowledge questions related to HPV and HPV-related oropharyngeal cancer and five self-perception HPV immunization administration questions were based on a validated questionnaire from Rutkoski et al. . Five adp < 0.05 was considered statistically significant.Frequencies and percentages were calculated for survey responses. Fisher exact tests were used to assess differences between groups  in proportions of a correct response for each of the 30 quiz questions. Blank responses found among thirteen participants and “not sure” responses were marked incorrect. Kruskal–Wallis and Mann–Whitney post hoc tests were used to assess differences between groups in attitude questions related to perceived responsibility, willingness, and understanding of HPV and OPC. For three post-training survey items that assessed improvement relative to pre-training directly, we used Wilcoxon signed rank tests to assess change among those who did not have the ability before the training session. For two post-training survey items assessing ability that was also assessed at pre-training, we used the Fisher exact test to compare outgoing D1–D4 pre- and post-participants as two independent groups. For purposes of comparison to pre-training assessment in these Fisher exact tests, the post-training responses that indicated their ability existed prior to training were considered as “strongly agree”. SAS version 9.4 software  was used to conduct analyses. A The Missouri Area Health Education Center HPV Immunization Project requested that the dental school provide an HPV education program for our dental students. Permission was given to JD to use the existing medically focused training program as a basis to develop a dental-specific one-hour training program. The training program included a PowerPoint presentation, informational handouts, and web addresses as resource materials. The PowerPoint presentation and handouts were sent to participants one day prior to the actual training.N= 171) or partially completed (N = 2) the survey. Of these, 111 (64.2%) were completed by outgoing students and 62 (35.8%) by incoming D1 students. The demographic characteristics of study participants are presented in All 228 students enrolled at the dental school were sent pre-training survey invitations, 173 (75.9% response rate) completed Student responses to the pre-training survey are presented in p < 0.048). In all these questions, fewer incoming D1 students answered correctly compared with outgoing D1–D2 and/or outgoing D3–D4 . About half of outgoing students and 29% of incoming D1 students correctly indicated the falsehood that “some types of HPV can cause HIV/AIDS”. Among outgoing students and incoming D1 students 25% and 11%, respectively, understood that OPC caused by smoking is more deadly than OPC caused by HPV. About 50% of outgoing students and 36% of incoming D1 students understood that HPV vaccines do not cause serious side effects. Between 25% of incoming D1 and 50% of D3–D4 knew 11–12 years is the recommended age for HPV vaccination in youth. Among outgoing students, in one question, more D1–D2 students knew that almost all cervical cancers are caused by HPV infection compared with D3–D4 students (73.6% (39/53) vs. 51.6% (30/58); post hoc p = 0.02). More consistent across groups, overall, only 14.5% (25/173) knew that the rate of HPV is not highest among women in their 30s and only 46.8% (81/173) knew that HPV vaccines do not have serious side effects. Knowledge of the association between HPV and OPC was well known with 86.1% (149/173) of students correctly indicating the link, and slightly less than half understood that people who have been diagnosed with HPV should not receive the HPV vaccine.In 14/30 questions, we found a significant difference between groups in the proportions of correct responses  at least somewhat agreed that both discussing and recommending HPV vaccination falls within the scope of a dental professional .p < 0.006). Half (86/171) of all students indicated it was extremely important as a dentist to provide counseling to parents about HPV and its link to OPC. Incoming and outgoing D1–D2 students tended to rate the importance of HPV vaccination counseling more highly than D3–D4 students (both post hoc p < 0.002). Slightly less than half of all students indicated they were very willing to administer HPV  or flu  vaccines. Incoming D1 students tended to be more willing to vaccinate patients for HPV or flu than D3–D4 students (both post hoc p < 0.001). The most common response was “agree” to items on perceived understanding of HPV and OPC. Incoming D1 and outgoing D3–D4 students tended to agree less with possessing the ability to describe the burden of HPV disease than outgoing D1–D2 students (both post hoc p < 0.01). Similarly, incoming D1 students tended to agree less with possessing the ability to explain the rationale for vaccinating youth than outgoing D1–D2 students (post hoc p = 0.02). No differences were found for year in school for other questions about perceived understanding related to HPV and OPC .The outgoing D3–D4 students tended to agree least with these two questions, and incoming D1 students had greatest agreement (both post hoc p < 0.001). After the training, including those who indicated they already possessed the ability, more agreement was found at post-training compared with pre-training for being able to define the importance of HPV vaccination for cancer prevention (post: 92% (131/143), pre outgoing: 83% (91/109); p = 0.004) and provide needed HPV vaccination information to parents (post: 91% (130/143), pre outgoing: 49% (53/109); p < 0.001).Of 165 outgoing D1–D4 students, 143 (86.7% response rate) completed the post-training survey self-assessing their ability to provide HPV-related education and counseling as a result of the training session. Responses are summarized in The purpose of the current study was to assess knowledge and attitudes about HPV and HPV vaccination for OPC prevention in first-year through fourth-year dental students. There were 14 questions where incoming D1 students were less knowledgeable than at least one of the outgoing groups. In the remaining 16 questions, there was not a statistically significant difference among the three groups. The willingness to administer the HPV vaccine successively decreased from incoming D1, to outgoing D1–D2, to outgoing D3–D4 students, who showed the least willingness to administer the vaccine. To our knowledge, our study was the first to investigate knowledge, attitudes, and intentions of dental students regarding HPV infection and vaccination across all 4 years of dental education.Knowledge about HPV’s link to OPC was known among most students in the pre-test survey and did not show a statistically significant association with dental school year. Over 85% of students could not confirm women in their 30s are not the population with the highest rate of HPV, and this also did not show a statistically significant association with dental school year. Student responses also indicated a misunderstanding of HPV infection sequelae: only a minority knew that HIV/AIDS cannot result from an HPV infection. These findings should concern oral healthcare professionals. Providing accurate information about potential consequences of HPV infection can improve patient confidence in dental providers and position dental professionals as trusted experts on HPV. Therefore, dental professionals need to be educated and prepared to counsel patients and parents about HPV, its link to OPC, and administration of the HPV vaccine as a primary prevention strategy.In terms of HPV vaccine knowledge, participants were less knowledgeable about serious side effects of the vaccine, the recommended age for vaccination, and the safety of vaccinating individuals with a past HPV diagnosis. These results should concern dental educators because they suggest that some students enter the dental workforce ill-prepared to provide accurate information to patients and parents about the HPV vaccination.Overall, students in the current study were unaware that OPC caused by smoking is more deadly than HPV-associated OPC. Patel et al.  found siThe majority of dental students in our study at least agreed that recommending the HPV vaccine to patients is within the scope of practice of dental professionals. Research suggests that parents of children with at least one dose of HPV vaccine had HPV vaccination recommended by a healthcare provider more often than parents of children who were not vaccinated . In our The majority of study participants strongly agreed that discussing the link between HPV and OPC was within the scope of practice of dental professionals. On the pre-training survey, less than half agreed or strongly agreed that they could adequately discuss the burden of HPV disease with patients or provide compelling enough information about the HPV vaccine to convince parents to vaccinate their children. Despite these results, a majority of students agreed that they were aware of the Centers for Disease Control and Prevention recommendations for HPV vaccination, the rationale for vaccinating youth aged 11–12 years, and the importance of HPV vaccination for cancer prevention.The current study did not investigate why students felt unprepared to convince parents of the value of HPV vaccinations. Torres et al.  reportedMost students in the pre-training survey were willing to administer the HPV vaccine in the dental office if properly trained. This finding is important for future dental practice. Although participants indicated for the most part that they were at least willing to train to administer the vaccine, Patel et al.  found deSurprisingly, we found a progressive decrease in willingness or strong willingness of dental students to administer HPV vaccines in the dental office as students progressed through dental school. Our study did not investigate the reasons for dental student unwillingness to administer the HPV vaccine; however, Kepka et al.  reportedFuture research could investigate the effectiveness of HPV training longitudinally throughout the four-year dental curriculum on student knowledge and intent to administer and manage HPV vaccinations and storage, tailoring the curriculum to potentially alleviate reluctance of students to administer the vaccines as they get nearer to graduation. Studies could also investigate how educators can maintain student focus on promoting HPV vaccinations to parents and patients. The recent Executive Order allowing dentists to administer the COVID-19 vaccine signals policymaker acknowledgment that dentists can provide vaccinations. We need to ensure that our dental students are leaving dental school willing to do so.The current study had several limitations. The students were from a single dental school, so results are not generalizable to other populations or institutions. Another limitation is that incoming D1 students completed only the pre-training survey. Future studies should consider training and surveying these students regarding intent-to-vaccinate for comparisons with current dental students. Further, asking students about their intentions on the pre-training survey and then linking their pre-training and post-training survey responses may more definitively indicate the effectiveness of the HPV training session.Participants nearer to graduation than those in earlier dental school years indicated they would be less willing to administer the HPV vaccine, despite showing sometimes more or no significant difference in nearly all knowledge scores about HPV or the HPV vaccine. Given the results of the current study, dental educators should evaluate their curricula for the timing and content of HPV training to ensure that graduating students are trained and willing to provide HPV vaccinations to reduce HPV infections and HPV-associated OPC. Future research should investigate the timing and content of different HPV-related curriculum models in dental schools for post-graduation willingness to administer HPV vaccines.


Acinetobacter baumannii and described the lytic capability and endolysin activity of the phage to evaluate the potential in phage therapy.Widespread misuse of antibiotics caused bacterial resistance increasingly become a serious threat. Bacteriophage therapy promises alternative treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated and characterized a novel, potent lytic bacteriophage against multi-drug resistant (MDR) A. baumannii isolates. Finally, HeLa cells were used to examine the safety of the phage.A novel phage, pIsf-AB02, was isolated from hospital sewage. The morphological analysis, its host range, growth characteristics, stability under various conditions, genomic restriction pattern were systematically investigated. The protein pattern of the phage was analyzed, and the endolysin activity of the phage was determined under the non-denaturing condition on SDS-PAGE. The optimal lytic titer of phage was assessed by co-culture of the phage with clinical MDR Myoviridae family. It can quickly destroy 56.3% (27/48) of clinical MDR A. baumannii isolates. This virulent phage could decrease the bacterial host cells (from 108 CFU/ml to 103 CFU/ml) in 30 min. The optimum stability of the phage was observed at 37 °C. pH 7 is the most suitable condition to maintain phage stability. The 15 kDa protein encoded by pIsf-AB02 was detected to have endolysin activity. pIsf-AB02 did not show cytotoxicity to HeLa cells, and it can save HeLa cells from A. baumannii infection.The morphological analysis revealed that the pIsf-AB02 phage displays morphology resembling the A. baumannii bacteriophage, pIsf-AB02. This phage showed suitable stability at different temperatures and pHs, and demonstrated potent in vitro endolysin activity. pIsf-AB02 may be a good candidate as a therapeutic agent to control nosocomial infections caused by MDR A. baumannii.In this study, we isolated a novel lytic MDR The online version contains supplementary material available at 10.1186/s12941-022-00492-9. Acinetobacter baumannii (A. baumannii) is responsible for many health care infections, particularly burn and wound infections .A. baumannii (MDR-AB02) was used as an indicator for bacteriophage screening of the sewage samples. The phages were isolated and enriched using the enrichment method . Then, 106 CFU/ml of A. baumannii (AB02) was added to each well, followed by adding the phage at different MOI . As a control, Hela cells were treated with 108 PFU/ml of the phage without the addition of A. baumannii. In a separate experiment, the cells were first infected with 106 CFU of AB02. After 2 h, the phage was added to the infected wells. After incubation for 24 h, the cells were washed twice with PBS, incubated with trypsin solution , and the number of living HeLa cells was counted using Neobar cell count and microscopic observation [We investigated the toxicity of the isolated phage to Hela cells. HeLa cell line (ATCC CCL-2) was obtained from the National Cell Bank of Iran, Pasteur Institute of Iran . The HeLa cells ; therefore, it was chosen for further study.MDR Caudovirales and family Myoviridae following the current guidelines of the ICTV .The morphology of pIsf-AB02 was examined by negative staining of the phage and observation under electron microscopy. The phage had an icosahedral head of 70 ± 10 nm and a tail of about 60 nm Fig. . The phaThe stability of the pIsf-AB02 to different pH, chloroform, and temperature was tested. The phage pIsf-AB02 lost its infectivity at pH 3 and 11, while pH 7 is the most suitable condition to maintain the phage. The phage was stable at different temperatures ranging from − 20 to 25 °C. However, the phage titer was slightly dropped at 50 °C and reduced dramatically at 70 °C. The activity of the virus was not affected by chloroform treatment . As shown in Fig. 8 CFU/ml to 103 CFU/ml in 30 min. Lower MOIs (0.1 and 0.01) decreased the virus titer to the same point in 1.5–2 h. The results indicate that although higher MOI reduced A. baumannii concentration more quickly, but is not necessary for lysis.The lytic activity of pIsf-AB02 was assessed by inoculating the phage to MDR-AB02. Different MOIs of the phage were inoculated into AB02 . The genome of pIsf-AB02 could be digested by ase Fig. . It was The results of pIsf-AB02 phage protein analysis in denaturing conditions showed nine structural protein bands in 12% SDS-PAGE, with a molecular weight ranging from 14.5 to 150 kDa. The most abundant proteins band in the gel were 100 kDa and 15 kDa. The major band was assumed to be the phage putative coat protein. The latter was predicted to be endolysin, which was confirmed with a non-denaturing condition Fig. .Fig. 8ThAt the time of release, the phages erupt the bacteria, causing the endolysin to flow out into the medium. Proteins of supernatant were concentrated and separated by SDS- PAGE under the non-denaturing condition as described before. The MDR-AB02 overlay on SDS-PAGE showed a clear band at 15 kDa Fig. .Fig. 9PhA. baumannii clinical isolates and showed that the pIsf-AB02 phage could infect and lyse 56.3% of the A. baumannii isolates (S1). The results demonstrated that phage was specific for the A. baumannii and did not affect Klebsiella, Pseudomonas, E. coli.Host range spectrum surveyed on forty-eight A. baumannii with a high titer of the lysate (1010 PFU/ml) were monitored for 7.5 h. Phage infection significantly decreased the A. baumannii culture turbidity in comparison to control. However, an increase in turbidity (OD600) was observed after about 4 h of culture incubation. This increase in turbidity was most probably due to the growth of phage-resistant bacteria   to survive as well as uninoculated controls. Although, the lowest cell viability was found at an MOI of 0.01. In bacterial control (cells inoculated with MDR-AB02 without phage treatment), all cells were completely killed by A. baumannii. Cells treated with the phage at an MOI of 10 (107 PFU), but not inoculated with bacteria, survived as well as the control cells (p > 0.05), indicating that high dose of pIsf-AB02 did not affect HeLa survival. The results showed that pIsf-AB02 eliminated bacteria and protected HeLa cells from immediate killing by A. baumannii AB02 bacteria.For the safety of phage therapy, HeLa cells were used to examine the cytotoxicity of the isolated phage in the presence of MDR-AB02. In 96-wells plate, different dilutions MOI = 10, 1, 0.1, 0.01 of pIsf-AB02 was added in the presence of 10ll) Fig. . Phage aIn another experiment, the cell viability was assessed by adding the phage 2 h post-infection of the cells with MDR-AB02. As shown in Fig. A. baumannii infections. The antibiotic sensitivity of 48 A. baumannii isolates revealed that the prevalence of MDR and pan-drug A. baumannii is increasing in the region, which is in agreement with other studies in different countries [The objective of our study was to demonstrate a new approach for using the bacteriophage to treat life-threatening MDR ountries , 32.A. baumannii isolate. It was able to form clear pinpointed plaques on MDR-AB02 lawns, indicative of strong lytic activity, and a wider host range among MDR A. baumannii clinical isolates  than our previously screened bacteriophages [. The major limitations of phage therapy are their narrow host range and the emergence of bacteriophage-insensitive mutants (BIMs); therefore, a large cocktail of phages is needed to improve therapy by extending the host range and reducing resistance and it is very important to isolate novel phages to enrich the phage supply.We isolated and characterized pIsf-AB02, a lytic phage from a clinical MDR iophages . The majMyoviridae based on its morphological characteristics. Regeimbal et al. in the United States also isolated a particular A. baumannii phage from sewage and stated that the phage had a shaft-shaped head along with a contractile tail and belonged to the Myoviridae family [Studying the isolated phage by TEM indicated that phage pIsf-AB02 should be assigned to the family of e family . Our isoe family , 34.A. baumannii phage from sewage was isolated and introduced through microscopic examination to the Myoviridae family. They also examined the phage stability at different temperatures, pH, and chloroform and showed that after a 24-h incubation of the phage at 37 °C, the potency of the phage remained unchanged and was stable in exposure to chloroform and normal pH [A. baumannii contaminations and or the treatment of A. baumannii infections. Phage pIsf-AB02 revealed impressive characteristics compared to the other phages. After a 24-h inoculation, the phage exhibited a steady state for chloroform. These results are consistent with the results of Kusradze [In a study by Kusradze et al., an ormal pH . BroadlyKusradze . ThermalKusradze , 36; howKusradze .A. baumannii isolates and a boarder range of temperature and pH stability [One-step growth curve analysis revealed a 30-min latent period, a 70-min lysis period, and a burst size of 120 phage particles per infected host cell. Compared with the isolated phage by Yang et al., pIsf-AB02 had a smaller burst size but a wider host range among the local tability , making 8 CFU/ml to 103 CFU/ml and maintained this concentration for about 2 h. However, the lower MOIs (0.1 and 0.01) decreased the virus titer to the same point in 1.5–2 h. The optimal titer of the phage in phage therapy is crucial. Despite the thought that the higher MOI of phage inoculation would get higher efficiency on waning the bacterium, the interaction between hosts and phages should be optimized. It might be possible that the high titer phage would occupy the receptor for phage and the bacterial lysis rate would not rise with an increase in MOI of phage [In 30 min, pIsf-AB02 with MOI of 1 reduced MDR-AB02 from 10of phage . Anotherof phage .A. baumannii.Herein, a high concentration of bacteria was applied to infect the HeLa cells to examine the protective efficacy of phages on the cells. The data showed that in the presence of the bacteria, pIsf-AB02 phage in higher MOIs didn’t affect on the survival rate significantly after a 24-h incubation in comparison to the control. On the other hand, the phage pIsf-AB02 did not have an adverse effect on the growth of HeLa cells, indicating that pIsf-AB02 is a suitable candidate for phage therapy. Further animal model experiments should be performed to confirm that phage pIsf-AB02 has a good protection on the animals infected by MDR Most double-stranded DNA phages accomplish host cell lysis through the holin-endolysin system. The similarity of bacteriophage endolysin genes is essential for structural analysis, which contributes to the potential of utilizing endolysin as an antimicrobial agent . The endPhage candidates for therapeutic application purposes should not harbor foreign genes, such as virulence or antibiotic resistance genes, integrases, site-specific recombinases, and repressors of the lytic cycle . Phages A. baumannii bacteriophage and evaluated the lytic activity of the phage against the isolated MDR A. baumannii. Furthermore, we assessed the efficacy of phage endolysin on MDR A. baumannii clinical isolates. Our findings support the potential application of the phage with the potent endolysin activity against MDR A. baumannii and suggest that this phage could be developed for the treatment of MDR A. baumannii infections.In this study, we isolated and characterized a novel lytic A. baumannii bacteriophage, pIsf-AB02. This phage showed suitable stability at different temperatures and pHs, and demonstrated potent in vitro endolysin activity. pIsf-AB02 may be a good candidate as a therapeutic agent to control nosocomial infections caused by MDR A. baumannii.In this study, we isolated a novel lytic MDR Additional file 1: Table S1.  The antibiotic sensitivity results and the spot test of pISF-AB2 phage, Figure S1. pH stability test, Figure S2: Thermal stability of pISF-AB2 phage.


Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11’s function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some “glutamine-addicted” cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H The membrane transporter, SLC4A11, is ubiquitously expressed in human and mouse tissues. Following the relatively recent discovery and cloning of the gene  in bovine corneal endothelial cells , but not [NH4+] or [H+]. If the [NH3] was held constant at each pH, the inward current was approximately the same, suggesting that SLC4A11 is activated by increasing [NH3] and not pH. These inward H+ currents were not Na+-dependent and were insensitive to EIPA [4Cl-sensitive inward currents in SLC4A11-transfected HEK cells that were, in contrast, mildly inhibited by EIPA. Using two electrode voltage clamps of oocytes transfected with mouse Slc4a11, Loganathan et al. [+ currents stimulated by NH4Cl, but concluded that SLC4A11 transports NH3 rather than NH3-H+. Myers et al. [+-independent H+ conduction, but concluded that SLC4A11 was activated by alkaline pH. Moreover, the H+ conductance of SLC4A11 was found to be steepest at pH 8.5 and it was found that mutants can shift the optimum pKa [+ conductance that can be either Na+ dependent or independent, is sensitive to NH4Cl, and is stimulated by alkaline pH. Zhang et al. [3/H+ cotransporter. However, this does not rule out SLC4A11 being an NH3 activated H+ transporter, as demonstrating NH3 net fluxes is difficult. The mechanism of an NH3 activation is unknown, but could possibly be caused by changing the charge of pore amino acid residues that could also be accomplished by alkaline pH. A more recent study by Kao et al. [+(OH−) conductive transport stimulated by alkaline pH. Ammonia-stimulated currents were also increased in alkaline pH. The shift in the reversal potential from NH3 suggested NH3-H+ cotransport was competing with H+(OH−) and the data, fitting a theoretical model of NH3-H+ and H+(OH−) interacting competitively within the transporter.The apparent enhancement of Hth NH4Cl  led to ath NH4Cl  demonstr to EIPA . Kao et easing NH, but nots et al. , also usimum pKa . In summg et al.  proposedo et al. , using S+ flux by the stilbenes, H2DIDS, SITS, and DIDS, which was also effective in increasing H+ transport of the R109H mutant. Zhang et al. [4Cl-stimulated H+ currents. Interestingly, SLC4A11 appears to confer a modest amount of membrane water permeability [One of the features of the SLC4 family of transporters is their interaction with stilbene derivatives. SLC4A11 binding to stilbenes has been useful in membrane fractionation studies . Whereasg et al.  found theability . Severaleability ,57. Howeeability . Further+ flux and the corneal endothelium pump by actively balancing pHi. SLC4A11 KO shows reduced lactate production and MCT4 expression in corneal endothelium, suggesting that there is some functional association [+ flux via SLC4A11 can proceed in either direction to balance pHi, according to Nehrke; however the electrochemical gradient favors H+ influx. A potential model . Glutamate can subsequently be converted to αketoglutarate by glutamate dehydrogenase (GLUD), and release a second molecule of ammonia. This occurs primarily within mitochondria, where αketoglutarate can then enter the tricarboxylic acid (TCA) cycle and contribute to the production of reducing equivalents that drive the electron transport chain, increase oxygen consumption, and produce ATP. Increases in αketoglutarate can also drive the TCA cycle in reverse, a process called reductive carboxylation. Here, the goal is to produce citrate which feeds into biosynthetic processes needed for proliferating cells. This is often found in carcinomas that express the requisite increases in TCA cycle enzymes that facilitate reductive carboxylation . Given tUsing cultured human corneal endothelial cells and mouse corneal endothelium, which highly express SLC4A11, Zhang et al. found a high expression of plasma membrane transporters for glutamine and other amino acids, GLS1 and GLUD, and a second isoform of glutaminase, GLS2, in corneal endothelial cells . Glucose+ into the inner mitochondrial matrix. WT and KO cells had similar oxygen consumption rates (OCR) when incubated in glucose alone. However, glutamine significantly increased OCR in WT, but not KO. OCR analysis indicated that glutamine induced a significant proton leak in WT that was absent in KO. Glutamine caused an increase in the NAD+/NADH ratio, indicating greater ETC activity, and increased ATP levels in WT, but not KO, consistent with the increased OCR. Mitochondrial reactive oxygen species (ROS) production was significantly greater in KO cells incubated in glutamine. In the absence of glutamine, mitochondrial ROS levels in KO cells were significantly lower than WT. The ROS levels paralleled the rate of apoptosis, which was highest in KO with glutamine, and lowest in WT and KO in glucose alone. The percentage of KO cells that were TMRE (tetramethylrhodamine) positive, indicating an intact MMP, dramatically decreased when incubated in glutamine, indicating ongoing mitochondrial damage. However, of the KO cells that were TMRE+ in glutamine, the staining intensity increased significantly with time, indicating MMP hyperpolarization. This suggested that glutamine catabolism in KO cells leads to increasing MMP hyperpolarization, causing increased ROS production and damage to mitochondria that then depolarizes. The damage appears to induce MTP opening  transporter. Whereas earlier studies suggested a Na+ dependence, most recent studies fail to show this [+(OH−) conductance and the model of NH3-H+ and H+(OH−) interacting competitively within the transporter. SLC4A11 is present at the plasma membrane, mitochondria, and potentially other organellar membranes. The N-terminal variants may have a role in specifying targeting SLC4A11; however, this is not the case for mitochondrial targeting. Ammonia production within mitochondria activates inner mitochondrial membrane SLC4A11 H+ uncoupling. Is this due to NH3  activation of the pore or does NH3 diffuse from the matrix across the inner membrane and then participate in coupled NH3-H+ transport (+ cotransport. SLC4A11 KO causes reduced expression of MCT4, lactate production, and glycolytic activity in general. In WT cells, lactate production is increased in the presence of glutamine, which is significantly curtailed in the SLC4A11 KO [3 produced from glutaminolysis activates plasma membrane SLC4A11, thereby facilitating lactate efflux, as envisaged in SLC4A11 functions as an electrogenic Hhow this ,53,54. Transport ? At the C4A11 KO . This mi


X1), oil volume (X2), and TTN amount (X3). The particle size and encapsulation efficiency percent (EE%) were selected as dependent variables. The optimal formulation demonstrated spherical morphology under scanning electron microscopy (SEM), optimum particle size of 116.3 nm, and high EE% of 83.2%. TTN-loaded nanocapsules improved photostability compared to its methanolic solution. The in vitro release study data showed that tretinoin was released in a sustained manner compared to the free drug. The ex vivo skin permeation study demonstrated that greater drug deposition into the epidermal region rather than the deep skin was observed with a gel containing TTN-loaded nanocapsules than that of drug solution, respectively. The skin irritation test revealed that the nanoencapsulation of the drug decreased its irritancy compared to the free drug. These results revealed the promising potential of cationic nanocapsules for topical delivery of tretinoinCationic nanocapsules represent a promising approach for topical delivery purposes. We elaborated on a novel formulation based on the cationic nanocapsules to enhance the pharmacodynamic efficacy, user compliance, and photostability of tretinoin (TTN). To achieve this goal, TTN nanocapsules were prepared by the nanoprecipitation method. In order to statistically optimize formulation variables, a Box-Behnken design, using Design-Expert software, was employed. Three independent variables were evaluated: total weight of the cationic acrylic polymer ( Tretinoin (TTN) is a first-generation retinoid with keratolytic and anti-inflammatory activities which is used topically in the treatment of various dermatological diseases such as photoaging, acne, and psoriasis –3. HowevEncapsulation of tretinoin with various delivery systems such as solid lipid nanoparticles , 7, lipoPolymeric nanoparticles have been considered promising carriers for cutaneous use . The maiAmong the polymers which are employed in the preparation of nanocapsules, the poly copolymer containing quaternary ammonium groups (Eudragit RS100) yields great interest due to its cationic charge . It seemGenerally, dermal application of aqueous nanocapsules onto the skin is difficult due to their low viscosity and they are easily removed. So, in order to improve their rheological properties, spreadability, and skin residence time, nanocapsules are incorporated into the semisolid vehicles , 20.ex vivo skin permeation study of TTN from the optimum formulation was compared with the TTN solution. Furthermore the draize skin irritation test was performed to assess the irritation potential of TTN-loaded nanocapsules when embedded in a Carbopol gel on the rat skin.The aim of this study was to develop topical TTN-loaded cationic nanocapsules to provide a slow release of drugs and higher permeability in the skin layers and enhance user compliance and photostability. Hence, optimal production parameters with optimum particle size and encapsulation efficiency were adjusted using the Box-Behnken statistical design. Moreover, the Tretinoin (TTN) was purchased from Olon S.p.A. . Eudragit RS100 was obtained from Degussa . Sorbitan monooleate (Span 80), polysorbate 80 (Tween 80), and caprylic/capric triglyceride mixture were supplied from KLK OLEO , and Carbomer 940  and triethanolamine were obtained from Fluka Chemical (Switzerland). HPLC grade acetonitrile was acquired from Duksan (South Korea). And all chemicals and solvents presented pharmaceutical or HPLC grades.X1), TTN amount (X2), and oil volume (X3). The encapsulation efficiency percent  and particle size  were selected as the dependent variables. The independent variables  and dependent variables are reported in A three-level three-factor Box-Behnken (BB) design was employed to statistically optimize the formulation variables for preparing TTN-loaded lipid-core nanocapsules (LCNC) in order to obtain high EE% and optimum particle size. Development and evaluation of the experimental design were performed using Design-Expert® software . A total of 15 experiments were run, 12 of which represent the midpoint of each edge of the multidimensional cube, and the remaining three are replicates of the cube's center point. Three independent variables were evaluated: total weight of the cationic acrylic polymer  were measured by photon correlation spectroscopy  after adequate dilution of an aliquot of the formulation in purified water at 25°C [Particle sizes and zeta potentials (n = 3) was assayed by diluting an aliquot of the sample in 25 ml acetone and submitting it to sonication for 30 min to extract the drug. Before injecting them into the HPLC system, the samples were filtered in a 0.45 μm membrane. To determine encapsulation efficiency, an aliquot of the samples was placed in a 10,000 MW centrifugal filter device  and the free drug was separated from the nanocapsules using the ultrafiltration/centrifugation technique at 8000 × g for 15 min. The EE% was calculated as the difference between the total and free concentrations of TTN, determined in the nanostructures and ultrafiltrate, respectively. TTN was quantified by High-Performance Liquid Chromatography (HPLC) on the Shimadzu system with a Shimadzu multiwavelength UV-VIS detector (SPD-20AV). The analysis was carried out at 25°C on a C18 column . The mobile phase consisted of acetonitrile, Milli-Q water, and glacial acetic acid (85 : 14 : 1), filtered through a 0.45 μm membrane filter  in the isocratic mode. The flow rate was 1.5 ml/min, and the detector was set at 355 nmThe total content of TTN in nanocapsule suspensions (n = 3) and predicted responses given by the software [The optimized formulation was obtained using the Design-Expert® software on EE% to reach the maximum value and on particle size to obtain the minimum value . The sugsoftware .n = 3). 500 μl of the samples was withdrawn at 0, 10, 30, 50, 80, 100, and 120 min. Then, the samples were extracted using a centrifugal filter device as discussed previously and the amount of the drug was assayed by HPLC. Also, as a control, the TTN-LCNC and TTN-M-S coated with aluminum foil (UV protection) were evaluated in the same way [To evaluate the stability of TTN-loaded NCs, it was exposed to an ultraviolet (UV) lamp . The 2 ml TTN-LCNC and 2 ml TTN methanolic solution (TTN-M-S) (in a 1 cm quartz cuvette) were exposed to UV radiation for 2 hours (h) at a fixed distance of 10 cm  with 49% of 2-propanol and 1% Tween 80® to keep the sink conditions. The samples, either tretinoin methanolic solution (TTN-M-S) or TTN-loaded nanocapsules, were placed in the dialysis bag . This system was kept under continuous magnetic stirring of 150 rpm. Aliquots of 1 ml were withdrawn at predetermined periods and replaced by the same volume of the fresh medium. The amount of tretinoin was assessed using HPLC. The experiment was conducted in triplicate.The appearance of the particles was evaluated using a scanning electron microscope (SEM)  . First, The NC suspensions were stored for 3 months at 25°C and 4°C, and all samples were protected from the light and kept in the dark all the time. The samples were withdrawn at 0, 30, 60, and 90 days and were assayed by HPLC. Also, particle sizes and PDI and zeta potentials were measured by photon correlation spectroscopy  after adequate dilution with purified water. The analysis was performed at 25°C .w/w[Hydrogels (HGs) were prepared using mortar and pestle by adding nanocapsule suspensions (10 ml) in 0.7-gram (g) Carbomer 934 (HG-TTN-LCNC) [w/w.w/v) [μm membrane and injected into the HPLC system [The pH values of the hydrogels were determined to be suitable for applied hydrogels onto the dermal region, using a calibrated pH meter , after dilution of 1 g of the samples in 250 ml water (4% w/v) . The rhew/v) , 24. TheMale Wistar rats weighing 240–250 g were used in all experiments. The animals were kept in a temperature-controlled room (25 ± 2°C) on a 12 h light-dark cycle and supplied with standard diet and water. The animals were habituated to the experimental room for 3 days before the tests. All of the experiments were done in accordance with the National Institutes of Health Ethical Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Hamadan University of Medical Sciences with the ethical code of IR.UMSHA.REC.1397.255. Animals were randomly designated in different treatment groups, and all experiments were blindly performed.2 diffusion area [μl of the receptor fluid was withdrawn and replaced with a fresh receptor medium. The concentration of TTN in each aliquot of the withdrawn receptor fluid was determined by HPLC as previously described [The study was conducted at 37°C using a Franz diffusion cell with a 15.0 ml capacity receptor compartment and a 2.54 cmescribed .ex vivo skin penetration study (after 24 hours), the rat skin mounted on the Franz diffusion cell was carefully taken off. The sample on the skin surface was carefully washed. Then, the cleaned rat skin tissue was washed thrice with Milli-Q water and dried on a lint-free cotton swab. The epidermis was separated from the dermis by means of heat application [μm), the filtrate sample was assayed using the validated HPLC technique [After completion of the lication . The sepechnique , 29.w/w) tretinoin on 4 skin surface points with the area of 4 cm2. After 24 h, the parafilm adhered on the skin surface was taken off and the skin surface points were observed for any visible change such as erythema  after 24, 48, and 72 h. The erythemal scores were reported from 0 to 4 according to Draize, where 0 means no erythema, 1 slight erythema, 2 moderate erythema, 3 moderate to severe erythema, and 4 severe erythema [In our study, skin irritation tests were performed in two healthy white rabbits (each 3-4 kg). The animal procedures were performed according to the written approval of the Ethics Committee, Deputy of Research and Technology, Hamadan University of Medical Sciences . The skin on both sides of bodies was shaved, and 4 points were marked on each side. 500 mg of all hydrogels containing tretinoin-loaded lipid-core nanocapsules (TTN-LCNC), tretinoin methanolic solution (TTN-M-S), blank lipid-core nanocapsules (HG-B-LCNC), and commercial gel was applied to the shaved surface of each rabbit, at a dose of 0.05% . The Box-Behnken response surface design and model fitting were accomplished by one-way analysis of variance (ANOVA) using Design-Expert® software (V.7.0.0). In this study, the comparison of two groups of data was performed using the two-sample independent Hvap of 32 kJ mol−1 or less could be completely removed under reduced pressure. Since acetone does not form any azeotrope with water and considering a part of the water was eliminated by evaporation at 40°C under reduced pressure, it can be concluded that acetone was completely removed from the water-acetone mixture [Primary studies were carried out to carefully select the most proper method for the preparation of TTN-LCN. The nanoprecipitation method was used in which the TTN can be loaded during NC formation by evaporating the organic solvent. Solvent removal parameters such as evaporation temperature, time, and speed were selected based on the characteristics of the prepared nanocapsules (such as size and PDI) and previous studies , 13, 31. mixture . So the The particle size, PDI, zeta potential, and EE% of the NCs (15 runs) are exhibited in A) and drug amount (B) on the EE%. The ANOVA test for the observed EE% data indicates that the quadratic model was significant and fitting for the data. The final equation in terms of coded factors was as follows:The ability of NCs to encapsulate significant TTN amounts is necessary for the targeted topical treatment for acne. Values of the EE are reported in p = 0.0002) of the concentration of TTN on the EE%. The increased amount of encapsulated TTN with increasing concentration could be due to the saturation of the organic medium with TTN that forces the drug to be encapsulated into NCs [During the preparation of TTN-LCNC, the TTN was introduced in the organic phase. So, TTN will probably be entrapped in the inner lipid core of the nanocapsules (LCNC). The equation reveals that there was a significant effect  and drug amount (C) on the particle size. The ANOVA test for the observed particle size data indicates that the linear model was significant and fitting for the data. The final equation in terms of coded factors was as follows:The particle size of the TTN-LCNC is reported in B, indicates that the oil amount had a synergistic effect on the particle size of the prepared NCs (p = 0.0001). The ANOVA results revealed the significant effect of oil amount components on the particle size (p = 0.0001). This may be attributed to the LCNC. This may have resulted in the increased diameter of the lipid core, and hence, particle size increased.NCs with very small sizes (less than 600 nm) delivered their contents into deeper layers of the skin . The posZeta potential is the measure of the overall charges of NCs and can be used to evaluate the stability of colloidal dispersions. When the zeta potentials of the nanoparticles are more than +30 mV or less than –30 mV, they are considered a stable colloidal suspension system due to electrical repulsion between particles . The valR2 was calculated as a measure of how well the model predicts a response value [R2 should be within approximately 0.20 of the predicted R2 to be in reasonable agreement [R2 values and adjusted R2 were in a reasonable agreement in both responses  was observed in both responses, as shown in se value . The adjgreement . Otherwiesponses . Then, tesponses . Therefop < 0.05). This means that the polymeric matrix could significantly screen out the UV radiation from the degradation of the TTN-LCNC.The TTN-LCNC degraded 29.49% ± 3.90 after 2 h of exposure to UV radiation, and the TTN-M-S degraded 89.75 ± 4.34% after the same exposure time . As it iThe SEM-based photographs revealed spherical polymeric selected TTN-LCNC , and froThe optimized formulation exhibited high stability in size and EE% with no signs of aggregation during the 90 days of storage at 25°C and 4°C temperatures .in vitro TTN release from the NC suspensions was investigated over 24 h. The results are reported in in vitro study, the TTN release from NCs was less than 10%, probably because of the slow release of TTN from NCs, while in the designated time period, more than 30% of TTN was released from TTN methanolic solution . As shown in the figure, in both formulations, the release of TTN was increased through the 24-hour incubation time and the final cumulative percent of TTN which was released from the NCs after 24 h was 80.5 ± 4.59% while the final cumulative release percentage of TTN from TTN-M-S was reported as 90 ± 3.64. In other words, the TTN release from NCs was significantly slower than the TTN-M-S after 24 h (p < 0.05). So, due to this release method, the prepared NCs are able to release the incorporated drug in a sustained manner.The p < 0.05). Drug delivery to the dermis layer was minimal, and TTN was not detected in the receptor compartment. This outcome was expected due to the high octanol/water partition coefficient of TTN [First, the ability of HG-TTN-LCNC to deliver TTN to the rat skin was evaluated and compared with the HG-TTN and HG-C. The retention results are provided in t of TTN . Howevert of TTN . Our worOne of the major side effects of the TTN is skin irritation (erythema), which strongly limits its utility and compliance by the patients. Ideally, the TTN delivery system should reduce or abolish these erythematic events. However, most of the marketed dosage forms  are not able to reduce the irritation caused by TTN topical therapy . The resin vivo skin deposition studies suggested that nanocapsules, due to their cationic charge, promoted drug deposition into the epidermal region rather than the deep skin, compared to drug solution. Moreover, the optimized formulation of TTN-loaded nanocapsules showed skin tolerability by the skin irritation study. In conclusion, the results revealed the promising potential of cationic nanocapsules for topical delivery of tretinoinIn the current work, topical TTN-loaded cationic nanocapsules were investigated to enhance their pharmacodynamic efficacy, user compliance, and photostability. The Box-Behnken (BB) design was employed to statistically optimize the formulation variables. The optimum nanocapsule formulation (F16) displayed spherical morphology, reasonable drug EE%, and optimum particle size with cationic charge. Nanocapsules provided an increased photostability of tretinoin, as well as a prolonged drug release. The performed