An association between HPV infection and progression to anal squamous intraepithelial lesions (ASIL) has been established, specifically in high-risk populations such as HIV-infected men. In this population, anal cancer is one of the most common non-AIDS-defining malignancies.A cross-sectional study to detect anal lesions and HPV infection was performed. Anal mucosa samples were collected from 56 HIV-infected men from Cuba. The cytological diagnosis was done according to Bethesda 2001 System. HPV DNA detection was determined by qPCR for six high-risk HPV types and end point PCR for low-risk HPV types (6 and 11). The end point PCR with nucleotide sequencing technique was achieved to detect other genotypes of HPV not included in the qPCR in those samples negative for HPV- 6 and 11 or negative for the six genotypes identified in the qPCR.p = 0.0006) and that the practice of receptive anal sex was a risk factor for anal HPV infection (p = 0.032).Cytological diagnosis identified 53 of 56 (95%) men with abnormal anal cytology. Among those, 26% (14/53) had atypical squamous cells of undetermined significance (ASC-US), 4% (2/53) had atypical squamous cells of undetermined significance cannot exclude high-grade lesions (ASC-H), 64% (34/53) had low-grade squamous intraepithelial lesions (LSIL), and 6% (3/53) had high-grade squamous intraepithelial lesions (HSIL). HPV DNA was detected in 89% (50/56) of men and 79% had at least one of the high-risk HPV types. HPV- 16 was the most common genotype (52%), while HPV-18 was the most frequently detected genotype in men with HSIL. We found statistically significant differences in the HPV viral loads with respect to the cytology results (This study shows a high prevalence of ASIL and high-risk HPV infections in the study group and is the first study showing the distribution of HPV genotypes in HIV infected Cuban men with abnormal anal cytology. This information may be of importance for local decision makers to improve prevention strategies, including the introduction of HPV vaccine in Cuba. While anal carcinoma is rare in the general population , it is oPersistent human papillomavirus (HPV) infection, especially high-risk HPV (HR-HPV) viral types such as HPV- 16 and HPV- 18, is associated with anal cancer and its precursor lesions, high-grade anal squamous intraepithelial lesions (HSIL) , 7. ThisLittle is known about anal HPV infection or anal cytological abnormalities in HIV-infected men in Cuba. The objective of this study was to investigate the distribution of HPV genotypes, and anal cytological abnormalities in HIV-infected men in Havana, Cuba.We performed a cross-sectional study to detect ASIL and HPV infection. Study participants were outpatients of the sexually transmitted diseases (STD) clinic at the Institute of Tropical Medicine “Pedro Kourí” and were recruited between April and August 2012. Those eligible to participate were HIV-positive males with a history of a previous or current STD, willing to give informed consent to an interview and tests for anal HPV infection and anal cytology. The inclusion criteria also included having an anal cytology useful for diagnosis.Prior to the sample collection, the anal canal was cleaned carefully twice using a saline- moistened cotton-flocked swab. The cytology specimens were collected by mean of a pre-scored brush , placed on a glass slide, stained with a Papanicolaou stain and classified using the Bethesda system criteria for evaluation of cervical cytological results , 12.TM . DNA was obtained using the QIAmp DNA Mini kit  following the manufacturer’s protocol.After the cytology, another sample was collected for DNA extraction. The brush was then placed in a 1 mL Specimen Transport MediumHPV DNA detection was determined by real time polymerase chain reaction (qPCR) for six HR- HPV types , as described below. HPV- 6 and 11 were detected using type specific end point PCR with L1 primers . The endThe methodology used in this study is based on a protocol previously published by Schmitz et al. in 2009  and it hPCR primers and corresponding TaqMan probes were used for HPV types −16, −18, −31, −33, −45, and −58 for target amplification of the LCR/E6/E7 regions of HPV genome. To identify and quantify viral load of those HR-HPV types, single PCR reactions were performed for each HPV type. To control for DNA quality, β-globin was amplified for each sample, in one of the reactions. Sequences of primers and probes are listed below (Table 6 and 10 copies/μL).HPV-16 and 18 standard curves were obtained from purified genomic DNA of SiHa and HeLa cell lines, respectively. HPV16-positive cervical carcinoma cell line SiHa (2 genome copies of HPV-16) and the HPV18-positive cell line HeLa (10–50 genome copies of HPV-18) were used to generate standard DNA for measurements of viral load  and low error values throughout 6 target DNA concentrations. The system had a lower detection limit of 10 copies for HPV-16 and other genetically related genotypes such as HPV- 31, −33 and −58. The lower detection limit for HPV-18 and other genetically related genotype, HPV-45, was also of 10 copies of viral genome. No cross reactions between HPV types and other DNA viruses were observed , 16.PCR was performed in a Light Cycler 1.5 platform . The PCR reaction comprises 5 μL of DNA (up to 50 ng), 4 μL of Quantitative PCR TaqMan Master Mix , 10 pmol of each primer and 1 to 5 pmol of each probe in a final volume of 25 μL. The initial denaturation step at 94 °C for 10 min was followed by 45 PCR cycles at 94 °C for 15 s, 50 °C for 20 s and 60 °C for 40 s each.HPV viral load was measured using absolute quantification. The viral load values were expressed as copies/μL. Cases with more than one HPV type were reported as multiple infections.2, 200 μM of each dNTP, 5 μM of MY primers, 2,5-U Taq Polymerase , and 100 ng of each DNA. Each PCR was performed with first denaturation step at 95 °C for 10 min, followed by 45 PCR cycles at 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min. A final extension at 72 °C for 10 min was included.The amplification reactions included a set of degenerated primers MY09/MY11 . Each amPolymerase chain reaction products of positive samples were purified using QIAquick® PCR purification kit . These were subsequently sequenced using 1 μL of 5 μM of MY09 and MY11 as the sequencing primers, 8 μL of sequence reaction mixture DTCS Quick Star Master Mix supplied with the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit , 5 μL of purified DNA and 6 μL of water to complete 20 μL of reaction mixture. The sequencing reaction consisted of 2 min denaturation at 96 °C, followed by 50 cycles of denaturing at 96 °C for 20 s, 20 s of hybridization at 50 °C, and 4 min of extension at 60 °C. Once the sequence reaction was concluded, it was purified, after the protocol described in the DTCS Quick Star Master Mix commercial kit (Beckman Coulter). The sequencing fragments were run on the genetic analysis system CEQ 8800 (Beckman Coulter). Finally, the sequences were edited and assembled using Sequencher, version 4.10 . Human papillomavirus genotype was determined using Basic Local Alignment Search Tool  and confirmed by manual phylogenetic analysis, using CLUSTAL-X and the neighbor-joining method in MEGA version 5  .p value of < .05.Data were processed using IBM SPSS Statistics 19. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, using univariate logistic regression, as estimates of the association of HPV infection and anal cytology results with potential risk factors. Comparisons between viral loads from groups of patients with different cytological classifications were made using the Kruskall-Wallis and Mann—Whitney tests. All statistical tests were considered to be significant at a A total of 56 eligible participants consented to be included into this study. The socio-demographic, epidemiological, and clinical characteristics of the participants are shown in Table Overall, HPV DNA was detected in 89% (50/56) men and 79% (44/56) had at least one of the six HR-HPV types that we sought. The most common types detected were HPV-16 (52%), HPV-11 (48%), HPV-6 (41%) and HPV-18 30%). Infection with multiple HPV types was detected in 71% of the participants (40/56),  men had abnormal anal cytology. Among those with abnormal cytology, 26% (14/53) had ASC-US, 4% (2/53) had atypical ASC-H, 64% (34/53) had LSIL and 6% (3/53) HSIL.HPV prevalence was remarkable high in both normal and abnormal anal cytology. All cases with normal anal cytology had HPV infection 3/3). Among those with abnormal anal cytology, 89% (47/53) were positive for at least one of HPV type and 74% (39/53) were infected with multiple HPV types. Among the HPV-positive cases with abnormal anal cytology, 87% (41/47) had at least one HR-HPV genotype and 55% (26/47) had more than two HR-HPV types. The most prevalent HR-HPV types among cases with abnormal cytology were HPV-16  and HPV-18 . HPV-16 was the major genotype in men with ASC-US, LSIL or ASC-H, followed by HPV- 33 in men with ASC-US, HPV-18 and 33 in men with LSIL and HPV-18 and 45 in ASC-H. HPV-18 was the most frequently detected genotype in men with HSIL, followed by HPV-16 and 58. In men with normal cytology, HPV-16 and 58 were the most prevalent types detected, (Table /3. Amongp = 0.0006). Those classified as negative and ASC-US showed lower viral load . In contrast, higher viral load was detected in cases with LSIL, ASC-H and HSIL  . Sexually transmitted diseases were also a risk factor for viral infection . Regarding HPV infection, according to age distribution, viral infection was not associated with age but a slight pick in prevalence for the 20- to 29-years old individuals was observed.Table p = 0.026; OR = 11.69 CI 95% 1.20-114.31). In addition, a positive diagnosis of STD, predominantly anal condyloma, was found to be related with an increased risk of ASIL/ASC-H .The population with ASIL/ASC-H was compared with those with normal anal cytology and ASC-US. A higher prevalence of ASIL/ASC-H was observed among those having oral/genital sex as a regular sexual behavior  of the participants had normal anal cytology.It is difficult to directly compare the results from different studies due to the various characteristics of the study participants including age, stage of disease, status of immune system, and history of HAART. Salit and colleges, in 2010, evaluated the prevalence of abnormal anal cytology based on a cross-sectional study of 401 HIV-positive MSM from Canada. They found the highest prevalence of LSIL (43%). In a study of 247 HIV-Infected MSM, Pokomandy et al. found a prevalence of abnormal anal cytology of 80.9% . In the Our results showed receptive anal sex and other STD, specifically anal warts as risk factors for HPV infection in the studied population. A past history of receptive anal intercourse and recent receptive anal intercourses are the most consistently reported risk factors for prevalent anal HPV infections . A recenAmong MSM, there are few data on age-specific HPV prevalence. In our study HPV detection was very high and viral infection was not associated with age. However, we observed a slightly high prevalence of anal HPV in men aged 20- to 29- years.The majority of studies found a high prevalence of anal HPV that did not decline with age . ContrarThe significance of the high incidence of HPV detection in this population is uncertain. One potential explanation for the high prevalence of anal HPV infection among MSM and the lack of association with age is that HPV infection in the anus is more persistent than in the cervix. However, the persistence of some HPV types may not reflect the true persistence but rather clearance of an HPV type followed by subsequent exposure or reinfection with the same type .In our study, higher levels of HPV viral load were associated with abnormal anal cytology. Tamalet et al. reported that HPV 16 viral load was an independent factor for abnormal anal cytology . Rodel aSome limitations of this study should be considered. The small sample size of this study limited the analysis when interpreting the prevalence data, mainly to perform statistical association analysis. On the other hand, even when anal cytology results were confirmed by biopsy using standard anoscopy and patients were treated properly, this study had no access to biopsy results and to the outcome of each patient, because they were attended at coloproctology consults from different hospitals.In this study we found for the first time in Cuba, a high prevalence of HR-HPV infection in anal mucosa of HIV infected men. Because a large number of cases have shown cytological abnormality, we suggest the possibility of including a screening for HPV detection and genotyping in anal cancer precursor lesions of HIV seropositive individuals. This would facilitate the clinical management of these patients in the presence of anal cancer precursor lesions, taking into account the increased incidence of this disease in this risk group. Moreover, the information reported here may be useful to consider the possibility of including HPV vaccination in Cuba.


The synthesis of new thiazole derivatives is very important because of their diverse biological activities. Also , many drugs containing thiazole ring in their skeletons are available in the market such as Abafungin, Acotiamide, Alagebrium, Amiphenazole, Brecanavir, Carumonam, Cefepime, and Cefmatilen.6, which reacted with dimethylformamide- dimethyl acetal in the presence of DMF to afford the unexpected thiazole derivative 11. The structures of the thiazoles 6 and 11 were optimized using B3LYP/6-31G method. The experimentally and theoretically geometric parameters agreed very well. Also, the natural charges at the different atomic sites were predicted. HOMO and LUMO demands were discussed. The anticancer activity of the prepared compounds was evaluated and showed moderate activity.Ethyl cyanoacetate reacted with phenylisothiocyanate, chloroacetone, in two different basic mediums to afford the thiazole derivative 11 has more electropositive S-atom than thiazole 6. The HOMO–LUMO energy gap is lower in the former compared to the latter. The synthesized compounds showed moderate anticancer activity.Synthesis of novel thiazole derivatives was done. The structure was established using X-ray and spectral analysis. Optimized molecular structures at the B3LYP/6-31G level were investigated. Thiazole derivative The online version of this article (10.1186/s13065-018-0420-7) contains supplementary material, which is available to authorized users. Currently marketed anticancer medications have increasing problems of various toxic side effects and development of resistance to their action. So, there is an urgent clinical need for the synthesis of novel anticancer agents that are potentially more effective and have higher safety profile. The synthesis of different thiazole derivatives has attracted great attention due to their diverse biological activities that include anticonvulsant , 2, anti6 was previously obtained by the reaction of ethyl cyanoacetate with phenylisothiocyanate and propargyl bromide in DMF-NaH [The thiazole derivative  DMF-NaH . The pre2CO3 or sodium ethoxide solution afforded only one isolable product. The isolated product was identified as (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene) acetate (6). Its structure was established from X-ray analysis . The suggested mechanism for the synthesis of thiazole 6 is outlined in Scheme The reaction of ethyl cyanoacetate with phenylisothiocyanate and chloroacetone in DMF-Ksis Fig.  24] and and2CO3 6 was confirmed using X-ray analysis  afforded the unexpected thiazole derivative 11  maps shown in Fig. The natural population analysis is performed to predict the natural charges (NC) at the different atomic sites  cell line in comparison with the anticancer drug vinblastine, using MTT assay A mixture of ethyl cyanoacetate  in sodium ethoxide (0.23 g Sodium in 10 ml of absolute ethanol) was stirred for 10 min. To this mixture, phenyl isothiocyanate  was added dropwise and the mixture was stirred for another 1 h. Chloroacetone  was added to the reaction mixture and stirring was continued for 3 h. Finally, it was poured on cold water and the solid precipitate that formed was filtered and recrystallized from DMF to afford the same product which obtained from method A, yield 65%.6  and DMF-DMA  in DMF (3 mL) was heated on a water bath for 1 h, then left to cool to room temperature. The precipitated solid filtered off, washed with EtOH and recrystallized from DMF to afford the thiazole derivative 11 in 82% yield, m.p. 260 °C; IR (KBr) vmax 1669 (C=O), 2189 (C≡N), 2928 , 3056  cm−1; 1H NMR : δ 1.26 , 2.88 , 3.16 , 4.21 , 5.28 , 7.43–7.56 ; MS m/z (%) 369 , 299 (0.98), 271(1.36), 98 (100), 77 (10.05), 70 (7.8). calcd. for C19H19N3O3S: C, 61.77; H, 5.18; N, 11.37. Found: 61.82; H, 5.21; N, 11.28.A mixture of thiazole 6 and 11 were obtained as single crystals by slow evaporation from DMF solution of the pure compound at room temperature. Data were collected on a BrukerAPEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα radiation, λ = 0.71073 Å at 100 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT. SHELXT [ F. CCDC 1504892 and 1505279 contain the supplementary crystallographic data for this compound can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.The thiazoles of . SHELXT , 31 was The X-ray structure coordinates of the studied thiazoles were used for geometry optimization followed by frequency calculations. For this task, we used Gaussian 03 software  and B3LYThe cytotoxic activity of the synthesized compounds was determined against Human Colon Carcinoma (HCT-116) by the standard MTT assay , 29.6) and its unexpected reaction with DMF-DMA gave (Z)-ethyl 2-cyano-2-(5-((E)-3-(dimethylamino)acryloyl)-3-phenylthiazol-2(3H)-ylidene)acetate (11). Optimized molecular structures at the B3LYP/6-31G level are presented. Thiazole 11 has more electropositive S-atom than Thiazole 6. The HOMO–LUMO energy gap is lower in the former compared to the latter. The cytotoxic activity of the synthesized thiazoles was evaluated and the results revealed that thiazole derivative 11 had more activity than thiazole derivative 6.Stereoselective synthesis of (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene) acetate  of thiazole 6. Table S3. Hydrogen-bond geometry  of thiazole 6. Table S4. The crystal and experimental data of thiazole 11. Table S5. Selected geometric parameters  thiazole 11. Table S6. Hydrogen-bond geometry  thiazole 11. Figure S1. The atom numbering scheme of the optimized molecular structures of the studied molecules. Table S7. The experimental and calculated geometric parameters of the studied molecules. Table S8. The natural atomic charges of the studied systems using B3LYP method.


Optimal decisions across extended time horizons require value calculations over multiple probabilistic future states. Humans may circumvent such complex computations by resorting to easy-to-compute heuristics that approximate optimal solutions. To probe the potential interplay between heuristic and optimal computations, we develop a novel sequential decision-making task, framed as virtual foraging in which participants have to avoid virtual starvation. Rewards depend only on final outcomes over five-trial blocks, necessitating planning over five sequential decisions and probabilistic outcomes. Here, we report model comparisons demonstrating that participants primarily rely on the best available heuristic but also use the normatively optimal policy. FMRI signals in medial prefrontal cortex (MPFC) relate to heuristic and optimal policies and associated choice uncertainties. Crucially, reaction times and dorsal MPFC activity scale with discrepancies between heuristic and optimal policies. Thus, sequential decision-making in humans may emerge from integration between heuristic and optimal policies, implemented by controllers in MPFC. Alhough humans often make a series of related decisions, it is unknown whether this is done by relying on optimal or heuristic strategies. Here, the authors show that humans rely on both the best heuristic and the optimal policy, and that these strategies are controlled by parts of the medial prefrontal cortex. For instance, when current conditions are bad, decision-makers need to determine whether to forage immediately, or to wait until conditions improve; if current internal energy resources are low, however, waiting may lead to starvation. Conceptually similar multi-step decision problems arise in many different real-world contexts, for example, in business decisions when investors have to balance immediate and delayed threats of bankruptcy.In biological scenarios, decision-makers should normatively evaluate the long-term consequences of their actions with respect to appropriate reward or cost functions. Foraging for food, for example, requires sequential decision-making to avoid starvation: foraging choices should ensure that current and future metabolic reserves remain within homeostatic boundaries. Humans in particular can use their quite sophisticated—but bounded—decision-making capabilities to take multiple future outcomes into account7. Decision-makers could simplify such quandaries by resorting to model-free heuristics9 and by restricting the set of considered options10 and actions11, in order to approximate optimal multi-step policies without full tree search. For example, foraging options’ momentary probabilities or magnitudes imperfectly signal expected starvation probability. A considerable literature has addressed how humans make choices on simple, economic gambles with one to three steps16, but it is much less well known how humans evaluate deeper sequences17.Sequential decision-making requires searching over a tree of probabilistic future states. During foraging, avoiding starvation depends on the success of current and future foraging attempts, on the internal energy state of the decision-maker, as well as on momentary and expected foraging opportunities. A model-based tree search should take all these variables into account—with more branches to be evaluated the longer the considered time horizon. These intricate computations may exceed the capacities of the human brain and entail costs in terms of opportunities foregone due to the passage of time19. Our results show that participants rely on a heuristic decision policy as well as on the optimal policy—both of which relate to blood-oxygen-level dependent (BOLD) signals in MPFC regions. The discrepancies between the two employed policies scale with reaction times (RTs) and dorsal MPFC activity.Here, we asked to what degree humans rely on optimal vs. heuristic decision policies, and how these are computed neurally. We hypothesized optimal policy computation in multimodal regions of the medial prefrontal cortex (MPFC) known to integrate economic decision variables and to evaluate prospective outcomes20  to explain participants’ choices, RTs, and fMRI data.We tested how well the optimal policy and ten different heuristic decision variables explained participants’ choices  were best explained by the a priori optimal policy with a horizon of five time steps , we found that protected exceedance probabilities did not decisively distinguish between two-variable models including the optimal policy, or the binary energy state heuristic, respectively . However, log-group Bayes factors provided decisive evidence for the same model as in the fMRI sample, including the best heuristic and the optimal policy  behavioral sample n = 1, we fouTaken together, model comparisons suggested that participants’ choices were often predicted by a heuristic policy, but additionally choices followed the normatively optimal policy.n = 28; PEP = 1.000; behavioral sample: n = 21; PEP = 0.986; see Supplementary Fig. t-tests against midpoint of choice proportion: fMRI sample: n = 28; absolute t-values > 5.2, all p’s < 10−4; behavioral sample: n = 21; absolute t-values > 1.9, all p’s < 0.065; Supplementary Table Using the optimal policy implies computing future states. We performed detailed analyses to determine how many days participants thought ahead in the task. Participants were incentivized to consider a time horizon of five days. In the main task they would often not finish five days (to enhance fMRI design efficiency), but they were instructed that their payoff depended only on a random subset of ten forests that would be completed outside the scanner. When comparing models with different horizons, we found that participants’ choices were indeed best described by a time-horizon of five days  with the optimal policy on a trial-by-trial basis and thus the two were related by design .Models of choice data indicate that participants used both a heuristic policy, i.e., the probability of foraging success, and the optimal policy. Consequently, we predicted that RTs should reflect the choice uncertainties associated with these two variables.  Indeed, RTs were slower when choice uncertainties were high  = 4.40; p < 0.001; p < 0.001; mean predicted choices according to the used policy did not differ between these trials; p > 0.4).Given that participants’ choices appear to use heuristic and optimal policy for making their decision, the question arises how these two are computed in relation to each other. As the heuristic is easier to determine than the optimal policy, we predicted that choices following the heuristic policy should be faster than those following the optimal policy. We identified the subset of trials in the fMRI sample in which the two policies made opposite prescriptions. More specifically, we computed the policies from the behavioral sample and applied these policies to the fMRI sample to select trials with opposite choice prescriptions. Within this subset , mean RTs for trials in which participants’ choices followed the heuristic policy were faster than for trials in which choices followed the optimal policy . This effect was present in addition to influences of choice uncertainties . To do so, we implemented a main general linear model (GLM) that included the variables from the winning choice model and the RT model as parametric modulators during the choice phase: the probabilities of foraging success, the value differences between foraging and waiting according to the optimal policy (time horizon of five days), their associated choice uncertainties, and the discrepancies in choice probabilities, as well as log-transformed RTs. Given the central role parts of the MPFC in decision-making, we specifically focus on this region in the presentation of our results. All described clusters are whole-brain family-wise error (FWE) corrected for multiple comparisons at x;y;z(MNI) = 8; 29; 50; t = 5.80), in bilateral intraparietal sulcus  = −42; −45; 51; t = 4.42; right: x;y;z(MNI) = 47; −42; 50; t = 5.37), and the left frontal pole  = −42; 47; −5; t = 4.28), among other regions  = 6; 33; 6; t = 6.27; Fig. The momentary probability of foraging success, i.e., the variable underlying the heuristic policy, showed a positive relation with BOLD signals in a posterior part of the dorsal MPFC  = 6; 50; 6; t = 4.53) and mid-cingulate cortex  = 2; 14; 29; t = 5.31; Fig. The optimal policy showed a positive relation with activity in perigenual ACC  = 9; 59; −2; t = 6.24), dorsal MPFC regions  = 3; 56; 33; t = 4.50) as well as to the inferior frontal gyrus  = 48; 35; −5; t = 7.37) and the posterior cingulate cortex  = 15; −27; 39; t = 7.63), among other regions  = 6; 42; 38; t = 4.81), and in the IFG  = 48; 26; −9; t = 5.88; Fig. Lower choice uncertainty of the optimal policy scaled with activity in DMPFC, extending into ACC  = −9; 36; 38; t = 8.25; Fig. x;y;z(MNI) = −11; 12; 3; t = 7.16; right: x;y;z(MNI) = 11; 20; −3; t = 7.66) and bilateral IFG  = −30; 26; −5; t = 6.17; right: x;y;z(MNI) = 50; 26; −11; t = 6.85).Crucially, as in our RT data, we found evidence for an integrated computation of the heuristic and the optimal policy: DMPFC activity correlated in a trial-by-trial fashion with the discrepancies between the two policies  = −47; 39; 6; t = 4.63), in a similar region as described above for the probabilities of foraging success according to the presented foraging options  = −42; 47; −5; t = 4.28). The values of optimal policy according to the chosen options’ values were positively related to the VMPFC  = 2; 63; 11; t = 4.76), in a similar region as described above for the value differences according to the optimal policy  = 6; 50; 6; t = 4.53). These findings corroborate that BOLD signals in the left frontal pole and in the VMPFC were associated with variables on which participants based their choices.In a fourth GLM, we specifically analyzed how the values of the chosen options related to BOLD signals  tracked the realized outcomes, that is the impact of participants’ decisions on their internal energy state  = 0; −26; 48; t = 9.01; Supplementary Fig. Finally, classic reward regionsTaken together, our results show that variables relevant for participants’ choices and RTs scale with activity in multiple brain regions involved in decision-making—in particular in ventral and dorsal MPFC areas.28. Importantly, choices in our task were also explained by the normatively optimal policy. Participants’ choices were therefore best explained by the combination of two metrics that constitute the end points of a spectrum between sloppy (but easy) and exact (but difficult) solutions.This study addresses the neural computations required to make sequential decisions during virtual foraging, when participants had to navigate above a lower homeostatic boundary over a number of consecutive steps with probabilistic outcomes. We show that humans rely both on an easy-to-access heuristic and on an optimal policy that integrates over probabilistic future states. More specifically, we demonstrate that participants took advantage of a model-free heuristic available at the time point of decision-making. This heuristic of relying on the probability of foraging success performed best overall in explaining participants’ choices among a large set of alternatives . This finding is in line with studies showing that participants often base their choices on the overall probability of winning in another type of sequential decision-making task30), such policies do not follow from the given task variables in an obvious way. Any such model would thus likely require higher complexity than the linear combination of the probability of foraging success and the optimal policy (or it would only apply to a less complex setting than the one investigated here). It is an interesting question for follow-up research whether on specific trial types (possibly under-sampled here) participants may have used a more (or less) complex model. Also, it appears possible that in a more (or less) challenging task, participants may abandon the optimal policy in favor of a combination of two or more heuristics.We did not find decisive evidence that any linear combination of two candidate policies and variables explained our choice data better than the probability of foraging success and the optimal policy. Although it is theoretically possible that participants use a yet unknown decision policy . Identifying the precise temporal requirements of information integration processes is an interesting and challenging avenue for future research.32. On the other hand, frontal pole, DMPFC, perigenual ACC and VMPFC play roles in several decision-making process19. Since participants were more likely to choose waiting rather than foraging when the probabilities of foraging success were small, the identified region of the perigenual ACC and VMPFC scaled positively with the relative value of “waiting” vs. “foraging,” as signaled by the heuristic. This finding may thus accord with the role of a similar region in a conceptually different sequential decision-making task: In the task used by McGuire and Kable33 participants had to adaptively decide how long to keep waiting for future rewards with different, uncertain timings. The temporal unfolding of the subjective value during different waiting periods was related to the VMPFC. In both tasks, “waiting”  trades off current (opportunity) costs against potential gains in the future. Still, the fact that our analyses also identify regions outside the VMPFC that scale with the value of foraging (notably in the DMPFC) constitutes a difference between our findings and those by McGuire and Kable33. Interestingly, the uncertainty of the heuristic variable was negatively (and not positively) related to BOLD signals in several regions, in particular in the posterior cingulate cortex, an anterior part of the VMPFC, dorsal MPFC, and IFG. Put differently, these regions showed a positive association with the “easiness” of making a decision according to the heuristic policy. Negative relations of an uncertainty metric have previously been identified a region of the posterior cingulate cortex, which was slightly more posterior than the cluster identified here34.Our fMRI data revealed that the heuristic variable, i.e., the probability of foraging success, was positively associated with BOLD signals in the frontal pole, the IPS, and a posterior part of the DMPFC. The same heuristic was negatively related to signals in a region of perigenual ACC extending into VMPFC. By definition, appropriate heuristics are related to the given task demands and therefore brain regions associated with any heuristic variable likely depend on the particular heuristic used. For example, the involvement of the IPS observed in the current study might be related to its role in processing numbers36. Indeed, optimal decision-making in our task bears considerably resemblance with model-based learning38 since both involve searching across a tree of probabilistic future states. Here, we demonstrate brain activity when participants evaluated decision trees with extended time horizons independent of the uncertainties arising during learning. In particular, we found that a dorsal region of the MPFC correlates negatively with the choice uncertainty of the optimal policy . This region seems to be especially well positioned to integrated different types of decision signals related to reward values and actions19. An intriguing possibility is that this region may generally be related to the uncertainties in recursive evaluations of a tree of probabilistic future states, which is a key feature for inferring optimal solutions in many realistic tasks.Multimodal integration regions such as MPFC and IFG were associated with the optimal policy and also the choice uncertainty of the optimal policy. These regions have often been observed in studies testing for brain activity related to model-based learning processes40. Thus, our finding relating discrepancies between the heuristic and optimal policies to a part of the DMPFC could potentially indicate that this DMPFC region becomes increasingly engaged when the computations of the two policies prescribe divergent choices such that progressive evidence accumulation and competition processes are required for making a decision.The discrepancies between the two employed policies showed a positive trial-by-trial relationship with a prominent cluster in the region of the DMPFC, extending into the pre-SMA and the ACC . The DMPFC cluster overlaps with regions classically associated with multiple types of decision discrepancies45. Our approach could be extended to include learning or information seeking such that the optimal policy would require reducing uncertainty about environmental states (cf. 46).Our virtual foraging task does not require exploring or inferring unknown, uncertain, or unobservable, states, which distinguishes it from learning tasks aimed at comparing explicitly signaled vs. previously trained values and tasks designed to assess model-based vs. model-free reinforcement learning4. In the present study, we address the underlying choice mechanism. We show that humans minimize the probability of virtual starvation via combining heuristic and optimal policies. In general, our delineation of heuristic and optimal policy computations informs a substantial body of behavioral work supporting notions of nuanced trade-offs between heuristic and optimal solutions49 or of fine-grained specifications of rationality51. Our behavioral findings corroborate recent proposals that the optimality of decision-making depends on cognitive limitations of the decision-maker as well as the energy and opportunity costs incurred during the decision process52, such that a theoretically optimal model-based system ceases to be optimal under some circumstances. Our fMRI results relate activity in the MPFC to the model-based tree search implicated in the computation of the optimal policy. Brain activity associated with the discrepancies between heuristic and optimal policies points towards a mechanism of integrated computation that may well generalize over many decision scenarios with multiple probabilistic states. Thus, our study paves the way towards better understanding the computations of the neural systems involved in making ecologically optimal choices in the face of complex multi-step decision-making scenarios.We have previously shown that formal models incorporating homeostasis maintenance can explain economic choices better than standard economic modelsParticipants were recruited from a student population via mailing lists of local universities. Twenty-one participants participated in the behavioral study : age = 25.8 ± 4.3 years). Thirty participants were recruited for the fMRI study. Two had to be excluded . This resulted in a final sample of 28 participants  for the fMRI study. Due to time constraints, one participant in the fMRI sample only performed eight out of ten sessions and another only performed nine out of ten sessions. Participants were paid a show-up fee  plus a variable amount (see Instructions and task). Both groups performed exactly the same task with the only difference that one group underwent MR scanning.The study was conducted in accord with the Declaration of Helsinki and approved by the governmental research ethics committee . All participants gave written informed consent using a form approved by the ethics committee.s above zero, i.e., the agent “dies from starvation” upon reaching the energy state zero at any time step. Here, the energy state can have discrete values equaling one to five energy points . At each time step t, or “day,” the agent can chose to “wait” and incur a sure loss cw (of one energy point) or it can “forage” in which case the agent probabilistically gains an amount g (of zero to four energy points) or incurs a cost for foraging cf (of two energy points). We denote the probability of foraging success as p . The probability of unsuccessful foraging and thus of incurring cf is q = 1−p. The maximum energy is capped; in the highest energy state the agent cannot gain more but simply stays in the highest state if foraging is successful.To probe sequential decision-making, we propose a toy scenario and a corresponding Markov decision process (MDP) for a hunter-gatherer or any foraging agent that aims at dynamically maintaining homeostasis over time. See Fig. The agent “lives” within a given “forest” in which all relevant variables are specified  or not (fq). These probabilities constitute the entries in the associated off-diagonals (depending on the magnitudes of g and cf).A core component of an MDP is the transition matrix between these different states ; i.e., it should minimize the probability of reaching zero energy points within a fixed and finite time horizon of n days when starting with the internal energy state s. In our case, the instructed finite time horizon n is always 5 days and the starting energy state s at the first day can be 2, 3, or 4 energy points. Participants were incentivized accordingly, i.e., they received a monetary payoff (for a random subset of forests), if their energy was above zero at day 5 and nothing otherwise. This corresponds to a simple implementation of a reward function within the framework of MDPs: All transitions to zero are associated with a “reward” of −1 and all other transitions with a reward of 0.How should the agent’s optimal policy look? The agent should minimize the probability of starvation pstarve and thus the optimal policy depend on the agent’s choice at the current time step t and the next n−1 time steps—in addition to the dependence on n and s. In our finite-horizon scenario, the starvation probability thus depends on the number of time steps t, i.e., days within a forest.Starvation probability A rough and basic intuition for a more or less prototypical optimal policy under such circumstances is that in many types of forests the agent should forage when the weather is good and wait when the weather is bad; unless waiting leads to sure death on the next time step. When the energy state is high enough so that starvation is impossible within the remaining number of days , the agent is indifferent between the two choice options.p (from 0.1 to 0.9 in steps of 0.1) and gains g (from 0 to 4 in steps of 1) for a finite time horizon of n = 5 days. In our MDP, the possible policies consist of the probabilities of foraging f in each energy state s, environmental condition (“weather”), and each time step t (with the probabilities of waiting w = 1−f). To derive optimal policies in our finite-horizon scenario, we used backward induction: That is, we started from the final time step  and calculated the values of the two choice options  for each state for that last and final time step. These values depend on the possible transitions from the respective states  or shorter  time horizon than instructed. Time horizons are flexible in the sense that it is assumed that participants start in a new forest with a given time horizon n and then reduce their horizon on each day by one. If the horizon has reached one , it will remain one. Supplementary Fig. The optimal policy thus depends on the time horizon considered. Our task was designed such that participants should use a time horizon of 5 time steps and participants were instructed and incentivized accordingly. Nevertheless, it is a possibility that participants use a different time horizon. Therefore, we calculated optimal policies for the following time horizons: Since the optimal policies binarize the value differences over the two choice options , they do not allow for variability in the decision process . We therefore used the continuous value differences between the two choice options as predictors of participants’ choices, RTs, and fMRI data. For brevity, we often use the term “optimal policy” to refer to the value differences between the foraging and waiting (according to the normative horizon of 5 steps in our task).All calculations were carried out in MATLAB.Participants received detailed written task instructions, in which the different task components were introduced step by step  or in the MR scanner (fMRI sample).Participants were incentivized to avoid starvation over five days in a forest. To strike a balance between design efficiency and the requirement to make participants integrate over a number of future time steps, the number of days in a forest followed an exponential distribution with a mean of 2.5 resulting in 40 days in 24 forests per session. Importantly, participants were instructed to always consider staying five days in a forest and were monetarily incentivized accordingly. In the end, we randomly selected one of the 24 forests for each of the ten sessions. To determine participants’ payment, they were again presented with the two weather types and their current energy state and completed the 5 days within these forests. For each forest in which they survived (regardless of the precise energy state) they received additional CHF 1.50.Of the total possible number of 400 days, participants in the behavioral sample had reached the starvation state on 22.2 ± 6.0 trials  and 21.1 ± 5.9 trials (fMRI sample). In the remaining trials they did not make a response within the time limit in 8.6 ± 10.7 trials  and 6.4 ± 8.6 trials (fMRI sample). This left 369.1 ± 13.0 trials  and 368.3 ± 18.8 trials (fMRI sample) for analyses.p; (3) momentary magnitude of foraging gain g; (4) expected value (EV) of foraging; (5) current continuous energy state s (ranging from 1 to 5 points); and (6) binary energy state (indicating whether waiting would lead to sure death or not). Since waiting leads to sure death when the continuous energy state is one, the optimal policy never prescribes waiting in these situations . Therefore, at a continuous energy state of one the prescriptions of the binary energy state variable are always in line with the optimal policy. We also included the following variables: (7) current weather type; (8) number of days past in a forest; (9) change between past and current energy states (within and across forests); and (10) a type of “win-stay-lose-shift” (WSLS) strategy, which prescribes foraging if the energy state increased in the past trial  and waiting if the energy state decreased . In other words, WSLS is a binarized version of the change between past and current energy states. Additionally, we tested for a myopic optimal policy: (11) the optimal policy according to a time horizon of 1 day (h-1).We used logistic regression models (implemented in the MATLAB function mnrfit) of the following form:mentclass2pt{minimp, as first predictor and one of the other policies as second predictor . Additionally, we explored whether choice uncertainties and discrepancy guided the use of the decision variables included in the winning model. Specifically, we tested the following interaction models  optimal policy for a horizon of 5 time steps prescribed foraging and the optimal policy for another time horizon prescribed waiting; or vice versa (Supplementary Table 54 (http://cran.r-project.org/web/packages/lme4/index.html). The independent variables in the mixed effects model were the probabilities of foraging success, the value differences of foraging vs. waiting according to the optimal policy (with a horizon of five days), the choice uncertainties of the two policies, and the discrepancies between the prescriptions of the two policies. Random effects for participants included a random intercept and random slopes for all variables. Since we had no hypotheses for interactions, we did not include any interaction terms as fixed- or random-effects. Significance levels of the fixed-effects were determined by performing log-likelihood tests, which compared the full model to models without the respective factor .2; slice thickness 2.8 mm; 0.5 mm gap between slices; slice tilt 0°). Imaging data were acquired in ten runs of 170 volumes each. The first five volumes of each run were discarded to obtain steady-state longitudinal magnetization. Field maps were acquired with a double echo gradient echo field map sequence, using 32 slices covering the whole head . Anatomical images were acquired using a T1-weighted scan .Data were recorded in a 3 T  MRI scanner using a 32-channel head coil. Functional images were recorded using a T2*-weighted echo-planar imaging (EPI) sequence . A total of 37 axial slices were sampled for whole brain coverage  T1 reference brain template. Normalized images were smoothed with an isotropic 8 mm full width at half-maximum Gaussian kernel.56 . We collected electrocardiogram (ECG), pulse oximeter, and breathing belt data during scanning. After quality checks, we used ECG and breathing belt data for noise correction in 19 participants, pulse oximeter and breathing belt data in two participants, and no physiological noise correction in six participants. The corresponding confound regressors as well as the six motion correction parameters estimated from the realignment procedure were entered as covariates of no interest. Regressors were convolved with the canonical HRF and low frequency drifts were excluded using a high-pass filter with a 128 s cutoff.We corrected for physiological noise using RETROICOR as implemented the MATLAB PhysIO toolboxThe three distinct phases of the task , the choice uncertainties of the two policies, the discrepancies between the prescriptions of the two policies, and RTs. A second GLM included participants’ choices (as a binary variable) as a parametric modulator in addition to the variables entered in the main GLM. A third GLM only included participants’ choices as a single parametric modulator. A fourth GLM included parametric modulators in terms of chosen (and unchosen) options (and not in terms of the presented options as in the first three GLMs). Specifically, this fourth GLM contained the a parametric modulator for the chosen values of the employed heuristic  and a parametric modulator for the chosen values according to the optimal policy  as well as parametric modulators for the corresponding unchosen values along with parametric modulators for participants’ choices and RTs.In all four GLMs, the forest phase was parametrically modulated by the current energy state and the overall starvation probability across five days. The outcome phase was parametrically modulated by the change in energy state.We report analyses in which regressors competed for variance . In the main GLM, the maximum average shared variance was below 0.5 for all combinations of regressors.t-test on contrast images from all participants. All reported clusters are family-wise error (FWE) corrected for multiple comparisons at p < 0.05 using the SPM random field theory based approach. The cluster-defining threshold was p < 0.001. At this voxel-inclusion threshold the random-field theory approach in SPM correctly controls the false positive rate57.We performed a second-level one-sample https://figshare.com/s/1a4d75cb4176a3fef040. The neuroimaging data that support the findings of this study are publicly available at https://neurovault.org/collections/3242/.The behavioral data that support the findings of this study are publicly available at Supplementary InformationPeer Review File


Background: Adolescent HPV (Human Papilloma Virus) vaccination is yet to be introduced as a mandatory program in Poland. Polish literature on factors associated with adolescent HPV vaccination is scant, despite the fact that uptake is one of the poorest in the European Union. Objectives: To assess HPV awareness and identify independent predictors for parental willingness to have their children vaccinated against HPV. Methods: All parents of first grade students from three selected high schools in Zgorzelec, Poland, who participated in parent–teacher meetings at the time the study was conducted, had their children unvaccinated regarding HPV, and who gave informed consent to participate were included. There were 600 first grade students; 9 were vaccinated against HPV. This left 591 parents who met the eligibility criteria; the response rate was 76.1%. Results: Awareness of HPV was reported by 55.3% of 450 parents ; 85.1% expressed their willingness to vaccinate their children against HPV; 31.3% identified HPV as a sexually transmitted pathogen, and 36.2% identified it as a risk factor of cervical cancer. Multivariable logistic regression analyses indicated that being employed , having positive attitudes toward vaccines , previous information about HPV , and concerns about the side effects of the HPV vaccine  were independent predictors of parents’ willingness to vaccinate. Conclusions: Attitudes regarding their child being vaccinated against HPV were positive among Polish parents, even though awareness and knowledge of HPV in this group were low. Most of the significant factors that influenced their willingness were modifiable, such as being informed about HPV and having positive attitudes toward vaccines. Future interventions should focus specifically on vulnerable subgroups, such as unemployed parents. Worldwide, an estimated 630 million persons are infected with human papillomavirus (HPV) . The preA number of health organizations and scientific societies, e.g., the WHO , the CDC (Centers for Disease Control and Prevention), the ECDC (European Centers for Disease Prevention and Control), and the AAP (American Academy of Paediatrics), recommend HPV vaccination for girls aged 11–12 years and a catch-up vaccination for those aged from 13 to 18 regardless of sexual activity, as well as persons who are not yet sexually active ,7. In reWithin the EU, Poland is one of eight countries where HPV vaccination is yet to have been made part of a free-of-charge mandatory form of immunization programme. Vaccinations must be purchased at primary health care sites. Three vaccines—Silgard, Cervarix, and Gardasil 9—are licensed in Poland for the prevention of cervical and other genital neoplasia as well as genital warts; the cost of a full series is high (ca. 300–440 USD per series) ,11.Over the last decade a variety of studies have explored adolescents’ and parents’ attitudes towards HPV vaccination, as well as the determinants of HPV vaccination uptake among adolescents in countries where the coverage in this subgroup is optimal or suboptimal ,14,15,16The decision to vaccinate against HPV is often made by the parents and is affected by their knowledge of the vaccine and attitudes about its use . TherefoA cross-sectional study was conducted from June 2013 to October 2014 among parents of high school students from the city of Zgorzelec with 32,332 inhabitants, located in southwestern Poland. At the time the study was conducted there were two phases of secondary education in Poland: lower secondary education  for pupils aged 13 to 16 and upper secondary education (high school) for students aged 17 to 19. The study participants were parents of first grade high school students from three high schools in the city which comprised all high schools in this area. There were 22 first grade classes in the selected schools, and each class consisted of 10–34 students. All parents of the abovementioned students who participated in parent–teacher meetings at the time the study was conducted, had their children unvaccinated regarding HPV, and who gave informed consent to participate were included. Parents who were not present during the first parent–teacher meeting were asked to complete an anonymous questionnaire during the following meetings.preintervention ;the immediate postintervention phase .The larger study involved analytical survey of parents before and after a school-based intervention program targeting parents and comprised two parts:The intervention used in parent–teacher meetings at schools consisted of a 30–40 min presentation about HPV and leaflets/brochures about HPV. This paper describes the results obtained from the first part of the study.A structured anonymous questionnaire was used as the main data collection instrument. It was designed by the study team using a literature review ,16,17,18Regarding HPV knowledge, eight items were rated as “true”, “false”, “don’t know”, with one point being given for a correct response. Questions were divided into three groups: symptoms/outcomes; transmission routes and risks; therapeutic methods. A scale was created to measure knowledge level from 0 to 8. A total knowledge score was then calculated for each respondent. HPV knowledge scores were set as follows: poor, 0–4 (less than 51% correct); adequate, 5–6 (51–75% correct); good, 7–8 (>75% of correct).The study was approved by the local Ethic Committee in Wrocław .p values < 0.05 were considered statistically significant.Categorical data were presented as frequencies with percentages and continuous data as means. Chi square test was used for two group comparisons of categorical variables; associations between continuous variables were assessed with the Mann-Whitney test. The variables of interest were as follows: age (≤42 years/>42 years), gender , place of residence , education level (basic–secondary/high), marital status (single/married–cohabitate), religion (religious/not religious), employment status , number of children in the family (≤2/>2), together with a cancer history in a family member/friend , received information about HPV (yes/no), knowledge about HPV and attitudes toward vaccines such as: vaccines are effective in disease prevention (yes/no-do not know), concern the child is at risk regarding serious consequences of HPV infection (yes/no-do not know), concern that HPV vaccination would make their children more likely to have sex (yes/no-do not know), and concern about the side effects of HPV vaccine (yes/no-do not know), associated with an outcome variable. The question “Are you willing to vaccinate your child with HPV vaccine?” was used to assess the potential willingness to vaccinate a child with HPV vaccine. Several multivariable logistic regression models were constructed using the stepwise backward elimination method to arrive at the most parsimonious model with independent predictors for parental willingness to vaccinate. Initially, all explanatory variables listed above were considered in the models, regardless of an association or a trend toward an association shown with the outcome of interest in the biivariate analysis. The Akaike Information Criterion (AIC) was used to find the model of the best fit . RegressThere were 600 first grade students in the studied high schools at the time the study was conducted; 9 were vaccinated against HPV. This left 591 parents who met the eligibility criteria; the response rate was 76.1%.The sociodemographic characteristics of the participants are presented in Almost half of the parents  had never heard of HPV. The most common sources of HPV knowledge were television (53.8%), internet (41.4%), and leaflets (32.4%), followed by a gynecologist (25.2%), a pediatrician (11.4%), and a nurse (11%); this was a multiple-choice question.p < 0.05).The parental knowledge regarding HPV is presented in Regarding parents’ attitudes toward HPV, 414 (92.0%) stated that vaccines are effective in disease prevention; 194 (43.1%) were concerned about possible side effects of vaccination, and 256 (56.9%) were not; 155 (34.4%) were concerned that vaccination would make their children more likely to have sex, 93 (20.7%) were not, and 202 (44.9%) were not sure; 169 (37.6%) considered their child as being at risk regarding serious consequences of HPV infection, 76 (16.9%) did not, and 205 (45.5%) were not sure.n = 178; 39.6%), “11 and less years” ; 19 parents (4.2%) stated “I do not know”. Regarding parental willingness to vaccinate their adolescent children against HPV, 383 (85.1%) were willing to have their children undergo vaccination, 19 (4.2%) had a negative attitude, and 48 (10.7%) were not sure. In the opinion of the parents, the best age to vaccinate a child for HPV would be as follows: “15 years or more” , “12–14 years”  reported that any decision would be significantly influenced by a physician’s recommendation, almost one-third  would be their own sovereign decision, and 8 participants would possibly base their decision on recommendations made by family/friends.p = 0.03 and 87.0% vs. 79.0%, p < 0.05, respectively) (p = 0.003) and those who had ever heard about HPV . The percentage of parents willing to vaccinate their child for HPV was significantly lower in the group concerned about the safety of HPV vaccine . Bivariate analyses revealed that more mothers and parents who were employed were willing to vaccinate their child for HPV  . The ratAs presented in p = 0.03), who had positive attitudes toward vaccines , and who had ever heard of HPV . Concerns about the possible side effects of HPV vaccine were associated with lower willingness  to have a child vaccinated. Of note, although one variable (age) was an insignificant  predictor, it was still selected by a stepwise procedure and remained in the model together with some other, significant variables.Regression analyses were performed to assess factors associated with parent willingness to vaccinate adolescents against HPV . AcceptaAwareness of HPV among the parents of the first-year high school students was low. The most common sources of their knowledge regarding HPV were television, internet, and leaflets. The overall knowledge about HPV was poor: only about one-third of parents identified HPV as a sexually transmitted pathogen or as a risk factor of cervical cancer. Nevertheless, the majority intended to have their children undergo vaccination against HPV. In multivariable modelling, parents who were employed, had positive attitudes toward vaccines, and had ever heard of HPV were more likely to have their children vaccinated. Those who expressed concerns about HPV vaccine safety were less willing to vaccinate their children.The awareness of HPV infection among parents is crucial as it may support vaccination in the case of their own adolescent children ,14,16. HThis report presents one of the few studies undertaken to evaluate the determinants of a willingness to vaccinate adolescent children against HPV that specifically considers HPV knowledge level within its analyses. Our results show a disturbingly low HPV knowledge level among parents of adolescent students. Only 19% of the studied population correctly stated that HPV infection may be asymptomatic. Almost two-thirds did not recognize HPV as a risk factor for cervical cancer, which is a much higher percentage than reported in the Chinese study (52.5%) but lower than the rate observed in the UAE (85.5%) ,14. ThisThe low awareness and poor knowledge regarding HPV observed in our study population may be associated with a number of factors. Firstly, the demographic characteristics of this study sample showed that 44.4% only had primary education. Although there was no difference in the knowledge level by parents’ education level observed in this study, results from some previous studies show that higher education among parents was significantly associated with a higher level of knowledge regarding HPV ,14,26. POur results indicate that parental attitudes toward vaccinating adolescent children against HPV were generally favorable. There are a number of previous surveys that examined this issue regarding both genders or exclusively daughters ,15,17,27Adolescents included in this study belonged to the first grade of a high school, being at the age of about 17 years. This age would make all the students eligible for the first dose of HPV vaccine 11–12 years). The observed mismatch between the actual, disturbingly poor vaccination uptake and an overwhelming majority expressing willingness to vaccinate seems confusing. One of the possible explanations of this finding could be a social desirability bias—the tendency of parents to answer questions in a manner that would be viewed favorably by teachers present in the classroom where the questionnaires were filled out. This could have resulted in over-reporting of “good behavior” (willingness to vaccinate an adolescent child against HPV) or under-reporting of “bad” or undesirable behavior, instead of choosing responses that are reflective of their true feelings  years. T. ComparaAn interesting study was conducted in the US among mothers who were nurses; the majority intended to vaccinate a daughter if she was 16 to 18 years of age, about a half intended to do so if she was 9 to 12 years . ContrarFactors associated with parents’ anticipated uptake of HPV vaccination for their daughters and sons identified in this study are complex and similar to those reported by other countries ,16,18. WWe also found that receiving information on HPV was associated with a two-times-higher chance of willingness to vaccinate a child. Our results support previous evidence related to this issue ,25,31,32In our study, 43.1% of parents were concerned about HPV vaccine safety. Multivariable regression analyses revealed that it was a negative determinant of parental willingness to vaccinate a child against HPV. This may be explained by the fact that our study population received information mainly from the mass media, which tend to report predominantly on speculated negative outcomes of vaccines in general and the HPV vaccine in particular. This result is in line with current literature ,14,15,16The present study did not identify a statistically significant association between parental HPV knowledge level and vaccine acceptance. This result is similar to findings reported in a US study  and a stHPV vaccine acceptance in this study was associated with parental employment status, and employed parents were two times more likely to vaccinate their children. Governmental support that would make HPV vaccinations free of charge or partly subsidized would greatly increase uptake, especially among unemployed parents and/or those living on low income. Interventions should be initially directed to those areas that suffer from especially high incidence rates of cervical cancer .Our study revealed that parents were not confident regarding the safety of the HPV vaccine. Therefore, interventions should concentrate on strategies which could increase confidence. Parents should be informed that it is a cancer-prevention vaccine, which can offer substantial benefits and might reduce the likelihood of serious outcomes . The widThe results of the study also showed that being informed about HPV was associated with greater odds of parental willingness to immunize their child. Although healthcare providers play an influential role in parents’ decisions to vaccinate their children against HPV, the results of the study show that providers are not yet fully promoting the vaccine. Therefore, gynecologists, pediatricians, and GPs should increase their role in the communication and delivery of vaccination for this specifically vulnerable group at visits made by adolescents and their parents.Clinicians should also consider informing the parents directly through messages by telephone, text, letter, postcard, or other media. Another tool used to provide information about HPV for the parents of adolescent children in Poland could be education by qualified experts during periodic parent–teacher meetings. As an example, Canada has begun to shift its attention to education funding for parents with regard to the prevention of HPV infections . AdditioThe study has a number of limitations. It was conducted in a small city; therefore, the findings do not necessarily apply to other students in the region or even the country, and further studies at national level would be of value. As mentioned in the Discussion section, there might also be response bias in the study: parents filled out the questionnaires in the presence of teachers, and thus may have given more sociably desirable responses ,28. HoweThe surveyed parents obtained information regarding HPV mainly from nonprofessional sources which may influence their low knowledge level; however, the majority were in favor of their adolescent child being vaccinated against HPV. In common with other countries ,15,18,19


Introduction: In 2014, it was reported that there was a backlog of an estimated 1.2 million claims nationwide at the United States Veterans Administration (VA). This ecological occurrence opened up a space for asking and answering some important questions about health service deficits (HSD) of US veterans, which is the focus of the research reported on in this paper. The purpose of this study was to ascertain if rural veterans were more likely to experience HSDs than urban military veterans after controlling for a number of covariates. Methods: Bivariate and multivariate data analysis strategies were used to examine 2014 Behavioral Risk Factor Surveillance System (BRFSS) survey data. HSD was the dependent variable. Results: Two multivariate models were tested. The first logistic regression analysis yielded that rural veterans had higher odds of having at least one HSD. The second yielded that rural US veterans in 2014 who had higher odds of having at least one HSD were: 18–64 years of age, unemployed seeking employment, living in households with annual incomes lower than $75,000, without a university degree, not part of a married or unmarried couple, a current smoker, and/or a binge drinker within the last 30 days. Conclusions: The study described here fills identified epidemiological gaps in our knowledge regarding rural US military veterans and HSDs. The findings are not only interesting but important, and should be used to inform interventions to reduce HSDs for rural veterans. Since 1990, the United States (US) has engaged in multiple major conflicts at distinct periods of time. Starting in 1990, the US was involved in the first Gulf War, termed Operation Desert Storm ,3.In 2014, it was reported that there was a backlog of an estimated 1.2 million claims nationwide at the United States Veterans Administration (VA) . Few wouHSD ,9,10 is An earlier study , analyziIn the US, a higher per capita proportion of military veterans come from rural rather than non-rural communities . US miliAccess to care has often been noted as an issue in rural communities for both veterans and non-veterans . The VA The purpose of this study was to ascertain if rural veterans were more likely to experience HSDs than urban military veterans after controlling for a number of covariates. The study described here sought to fill identified epidemiological gaps in our knowledge regarding rural US military veterans and HSDs. Among the epidemiological gaps are whether or not rural veterans have greater odds of experiencing HSDs in comparison to urban veterans. Furthermore, we do not know if the VA has successfully bridged healthcare access disparities for rural veterans, reducing the possibility of HSDs as we have defined them. Analyzing population-based, non-VA, non-medical records or data for veterans might better identify the population of US military veterans (by specific characteristics) who may have HSDs, thus providing direction for health systems interventions aimed at solutions for reducing HSDs.To answer the research question, 2014 BRFSS data were analyzed using bivariate and multivariate techniques. BRFSS is a random digit telephone survey that is a collaborative project of the Centers for Disease Control and Prevention (CDC) and all US states and territories. The survey measures several behavioral risk factors and disease states in the non-institutionalized US adult population aged 18 through 99 years. A complex multi-stage sampling approach is used by BRFSS and subsequently a weighting factor is calculated for application to the data in order to ensure that they are representative of the US population based on the most recent census data. A more in-depth description of the data weighting process can be found elsewhere [Are You a Veteran? were used. All respondents selecting yes were considered military veterans.BRFSS collects information from individuals on health risk behaviors, preventive health practices, chronic conditions, and healthcare access primarily related to chronic disease and injury. BRFSS is composed of core questions that must be asked of every survey participant, as well as optional modules that may be chosen by individual states and asked only of the survey respondents from the participating state(s). To identify veterans for this study, responses to the question: don’t know and refused. Re-coded variables were: age, employment status, marital status, deferment of medical care within the past year because of cost, timing of last routine medical check-up, health insurance status, personal healthcare provider, education attained, annual household income, binge drinking, smoking status, body mass index (BMI), arthritis, depression in lifetime, diabetes, and geographic locale. All variable re-codes were undertaken for clarity of factors for analysis and ease of interpretation. For instance, in BRFSS, marital status has nine categories. These were re-coded into two categories: Married or Part of Unmarried Couple, and Not Married or Part of Unmarried Couple, because these two categories encapsulate the concepts of interest. Likewise, education had nine original categories that were reduced to three through re-coding. The re-coded categories represent the most meaningful ones for this analysis. Age was re-coded into two categories: 18–64 years of age and 65 years and older. This re-code was purposive, since in the US persons 65 years of age and older are covered by Medicare regardless of their military status.For analysis, a number of variables were either re-coded or computed. Re-coding for the most part entailed collapsing response categories and removing the response categories of The geographic locale variable was determined using the metropolitan statistical area (MSA) variable provided in the BRFSS database. MSA is comprised of geographic entities delineated by the Office of Management and Budget (OMB) for use by Federal statistical agencies in collecting, tabulating, and publishing federal statistics. The MSA variable was recoded into the dichotomous categories of rural and urban. Rural veterans were defined as people living either within an MSA that had no center city or outside an MSA. Urban veterans included all respondents living in a center city of an MSA, outside the center city of an MSA but inside the county containing the center city, or inside a suburban county of an MSA.Three variables were computed from other variables in the BRFSS database. These were race/ethnicity, chronic disease, and HSDs. Race/ethnicity was calculated from participant responses to two separate survey questions—one regarding race and the other regarding Latino/Hispanic ethnicity. All race/ethnicity categories were computed as mutually exclusive entities. For example, all respondents coded as Caucasian chose white as their racial classification, likewise, black for African American, etc. If a respondent identified themselves as Hispanic or Latino they were classified by that ethnic category regardless of any additional racial classification. The category of Other/Multiracial was also calculated.The chronic disease variable was a composite variable calculated from factors from four separate variables—have arthritis, have diabetes, have lifetime depression, and obesity, as measured by the calculated BMI. These were chosen because they are common and age prevalent. Having at least one of these conditions was coded as at least one chronic condition, although this did not preclude respondents from having more than one of these chronic conditions.As mentioned in the introduction, the variable HSD was computed from the factors of four different variables: no routine medical exam, no primary care provider, no health insurance, and/or deferring medical care because of cost, all within the last 12 months. Having at least one of these was coded as at least one HSD; this did not preclude respondents from having more than one of these. The variable HSD was the dependent variable for the study.p < 0.05. The Institutional Review Boards (IRBs) of the researchers’ institutions recognize that the analysis of de-identified, publicly available data does not constitute human subjects research as defined in federal regulations, and as such does not require IRB review. Human subjects review was not sought nor received.For bivariate analysis, since all of the independent variables were categorical, either a chi square or an odds ratio was computed as the test statistic. For the multivariate analyses performed, an adjusted odds ratio was the calculated test statistic. All analyses were performed on weighted data as is recommended by the CDC. The weighting, calculated by the CDC, uses the most recently available census data to provide a stratified representation of the nation’s non-institutionalized population. Only findings from weighted analyses were considered valid. All analyses were performed using SPSS version 24  with alpha set at The results of the first multivariate model tested are displayed in This study sought to ascertain if the geographic locale of US veterans impacted their odds of experiencing at least one HSD. More specifically, we were interested in determining if rural residency of veterans was an independent risk factor for HSDs after controlling for a number of possibly contributing covariates. A number of notable findings emerged from this study, the first of which was that rural veterans had higher odds of having at least one HSD when compared to their urban counterparts. Furthermore, our results yielded that rural veterans ages 18 to 64 had greater odds of experiencing at least one HSD. This second finding was not dissimilar to that regarding all US veterans regardless of geographic locale. Our results also revealed that rural veterans with incomes less than $35,000 also had greater odds of experiencing at least one HSD, as did those without a university degree. Rural veterans who were current smokers and who were binge drinkers in the past 30 days also had higher odds of having at least one HSD.These findings provide an epidemiological snapshot of rural US veterans experiencing at least one health service deficit in 2014. HSDs are not a small issue, given the difficulties of the VA in processing claims and providing healthcare for veterans that came to a head in 2014 . Rural rThat younger veterans had greater odds of having at least one HSD indicates that these are most likely veterans who have served in the Gulf-war era and the more recent Middle Eastern conflicts. All of the HSDs may be an issue for these veterans. For instance, there may be a common misperception that veterans are entitled to receive lifetime medical insurance and care. However, coverage for medical services is not guaranteed for all military veterans regardless of combat status . While iSmoking and binge drinking are health-related risk behaviors. Their strong association with HSDs and rural veterans is worth noting and addressing in any interventions focused on reducing HSDs for this population. It may be the case that binge drinking is indicative of some measure of self-medication. Furthermore, engaging in these risky health behaviors may indicate that other similarly risky behaviors not measured in this study are also practiced.Lower income and less educated rural veterans are a more vulnerable group of veterans. That these characteristics are also strongly associated with HSDs should be taken into consideration when developing any outreach or interventions aimed at reducing their HSDs.It is also interesting and notable that African American race was protective both generally and in the instance of rural residency for HSDs. It may very well be the case that, in contrast to their other race and ethnicity counterparts, African American veterans have applied for, received, and used the healthcare benefits they are eligible for. These findings bear further examination.Several potential limitations to this study should be noted. First, the survey is based on telephone-derived data and may lack representation because those who could not be reached by phone could not participate in the survey. For instance, persons of lower socioeconomic status may have been excluded because of poorer phone access. Widespread use of answering machines and caller identification now allow people to filter their phone calls, potentially leading to a passive refusal to participate in surveys such as the BRFSS. Nevertheless, call filtering is beyond the control of survey administrators and the vast majority of US residents live in households with telephones, which minimizes the bias of lack of phone access. Additionally, US cell phone numbers are now included in the pool of phones contacted for the survey ensuring the widest possible net being cast. A second limitation is that the survey used close-ended questions, which limit participants’ options to fully explain response choices. Nonetheless, the survey questions were worded such that the answer choices covered a wide range of response possibilities. A third, and related, limitation is that the answers are self-reported, which introduces the possibility of recall bias on the part of the survey participants. In addition to recall bias with self-reported data on health issues, there is the possibility of either under-reporting or over-reporting health concerns. Fourth, geographic locale is not provided for US veterans residing in Guam, Puerto Rico, or the US Virgin Islands. This may result in an under-reporting or under-identification of rural veterans. Finally, we were unable to identify a number of potentially important characteristics of the military veterans including combat status, location of services, deployment status, and length of active military service. Despite these limitations, there are a number of strengths worth noting. This is a population-based study analyzing data representative of the US population of veterans—rural and urban alike. Moreover, the data are surveillance data collected separately from any medical records or VA collected data.The study described here fills identified epidemiological gaps in our knowledge regarding rural US military veterans and HSDs. Rural veterans have greater odds of having an HSD when compared to their urban counterparts. A major strength of the study is that non-medical and non-VA data were analyzed. The findings are not only interesting but important and should be used to inform interventions to reduce HSDs for rural veterans.


E6/E7 oncogenes plays a key role for HPV-linked oncogenesis. It recently has been found that low oxygen concentrations (“hypoxia”), as present in sub-regions of HPV-positive cancers, strongly affect the interplay between the HPV oncogenes and their transformed host cell. As a result, a state of dormancy is induced in hypoxic HPV-positive cancer cells, which is characterized by a shutdown of viral oncogene expression and a proliferative arrest that can be reversed by reoxygenation. In this review, these findings are put into the context of the current concepts of both HPV-linked carcinogenesis and of the effects of hypoxia on tumor biology. Moreover, we discuss the consequences for the phenotype of HPV-positive cancer cells as well as for their clinical behavior and response towards established and prospective therapeutic strategies.Oncogenic types of human papillomaviruses (HPVs) are major human carcinogens. The expression of the viral  E6 and E7 oncogenes play a central role both for the induction of malignant cell transformation and for the maintenance of the oncogenic phenotype of HPV-positive cancer cells. Recent data indicate that hypoxia has profound effects on the crosstalk between the HPV oncogenes and their host cell, with implications for the malignant phenotype of HPV-positive cancer cells and for their clinical behavior.Approximately 50–60% of solid tumors exhibit pronounced hypoxic regions (commonly defined as oxygen concentrations below 1.5–2%) . HypoxiaIn principle, two major forms of hypoxia can be differentiated in solid cancers. Firstly, chronic hypoxia occurs in a time frame of hours to days or weeks. This form of hypoxia is primarily caused by diffusion limitations, e.g., due to enlarged distances >70 µm ) between0 µm  betThe poor clinical prognosis of hypoxic tumors could, on the one hand, mechanistically be due to hypoxia-linked processes that support the clonal evolution of more malignant tumor cells. These processes include (i) the induction of genetic instability through the downregulation of DNA repair mechanisms  and incr2 . Although called “normoxia”, these conditions do not reflect the median O2 content of normal tissues (“physoxia”), which lies around 5–6% O2 in most organs, with some exceptions [2 concentrations of less than 2% [Notably, although the pronounced effects of hypoxia on cancer cell biology are well established, they are not taken into account in standard cell culture experiments, where cells are usually incubated at approximately 20% OAt least 20% of the total cancer incidence in humans is attributable to infections . In thisTP53 and RB1 genes [TP53 and RB1 genes, the inactivation of the p53 and pRb pathways by the HPV oncoproteins is reversible. In particular, the inhibition of E6/E7 expression in HPV-positive cancer cells leads to the reconstitution of p53 and pRb signaling and to the induction of cellular senescence [In the process of HPV-induced carcinogenesis, the viral E6 and E7 oncoproteins target crucial tumor suppressor pathways for functional inactivation. For example, E6 forms a trimeric complex with the cellular ubiquitin ligase E6AP (E6-associated protein) and the p53 tumor suppressor protein , eventuaB1 genes ,27. Thusnescence ,30,31,32nescence .2 concentration of only 1.2%. HNSCCs appear to be only slightly better ventilated, with median O2 concentrations between 1.3% and 1.9% [Consequently, it is widely assumed that HPV-positive cancer cells are “oncogene addicted”  in that and 1.9% ,35. The 2) on HPV-positive cancer cells [A recent study investigated the effects of chronic hypoxia (1% Oer cells . It was E6/E7 silencing is linked to the reconstitution of the anti-proliferative p53 (and subsequently the p53-mediated stimulation of the negative cell cycle regulator p21) and pRb pathways [E6/E7 silencing by RNA interference (RNAi) [What is the mechanism underlying the discrepant phenotypic responses following E6/E7 repression in normoxic and hypoxic HPV-positive cancer cells? In normoxic HPV-positive cancer cells, the induction of senescence upon pathways ,37. Thispathways . More repathways ,39. Thise (RNAi) . CollectE6/E7 is silenced in HPV-positive cancer cells. This observation is also remarkable in the light of previous studies, which show that the HPV E6 protein stimulates mTOR signaling upon ectopic expression [The anti-senescent effects of the mTOR inhibitors in normoxic HPV-positive cancer cells also indicate that the mTOR pathway remains active in these cells despite strong E6/E7 downregulation. In line with this, and indicative for sustained mTOR signaling, the amounts of phosphorylated mTOR downstream targets are not  or are only partially (phospho-4E-binding protein 1) reduced when pression ,41, inclREDD1 or TSC2 expression by RNAi leads to the stimulation of mTOR signaling and to the emergence of senescent HPV-positive cancer cells under hypoxia [Importantly, the mTOR pathway is strongly repressed under hypoxia in many cell systems , includi hypoxia . CollectBesides surgical excision, RT and radiochemotherapy (RCT) play central roles for the clinical management of HPV-positive cancers, such as cervical carcinomas and HNSCCs. CT alone usually has only limited therapeutic efficacy, and often is applied as a palliative treatment when surgery or RT are not possible, e.g., in patients with recurrent or metastatic disease ,45,46. N2 is required to manifest the DNA lesions that are induced by ionizing radiation [MDR1 (multidrug resistance 1) gene [In particular, hypoxia is considered to be a major obstacle for the therapeutic efficacy of RT, mainly due to the fact that Oadiation ,48. As aadiation ,48. The  1) gene ; and (ii 1) gene . Thus, t 1) gene , but als 1) gene ,52,53. H 1) gene .Besides these classical treatment regimens, there are intense efforts to develop novel therapeutic strategies targeting E6/E7 in HPV-positive cancers. The theoretical basis for most of these approaches is the conception that the HPV oncoproteins are regularly expressed in HPV-positive cancer cells and that they play a crucial role in the maintenance of their malignant phenotype. Accordingly, antigens derived from the viral oncoproteins are believed to represent attractive targets for immunotherapy of cervical cancer or HPV-positive HNSCCs, since HPV-positive cancer cells would be unable to downregulate E6/E7 expression as an immune evasion mechanism ,55. HoweFurthermore, the rapid and efficient senescence response that is observed upon E6/E7 repression in normoxic HPV-positive cancer cells raises the possibility that inhibitors of E6/E7 expression or function possess therapeutic potential for the treatment of HPV-positive tumors ,31,32,60In view of the potentially pro-tumorigenic effects of the SASP of surrounding stromal cells, it thus might be beneficial to induce senescence selectively in tumor cells. In principle, this specificity could be achieved by interfering with E6/E7 expression or function, since these therapeutic targets are not present in normal (HPV-negative) cells. Unfortunately, however, hypoxic HPV-positive cancer cells would be expected to resist a pro-senescence therapy that is based on E6/E7 inhibition, since the expression of the therapeutic targets is blocked and pro-senescent mTOR signaling is impaired .Magnetococcus marinus strain MC-1 that can transport drug-containing nanoliposomes into hypoxic tumor regions [Clostridium novyi-NT that spreads to and destructs hypoxic cancer areas upon intratumoral injection [Yet, it should be emphasized that these considerations do not preclude a therapeutic use of prospective E6/E7 inhibitors, since they would be expected to act in a pro-senescent way in non-hypoxic HPV-positive cancer cells where the therapeutic targets are expressed and mTOR signaling is active. The inhibition of E6/E7 could thus be combined with treatment strategies currently under development that aim to attack hypoxic cancer cells . These i regions , or Closnjection .Notably, the phenotype of hypoxic HPV-positive tumor cells may not only provide therapeutic resistance, but could also be a risk factor for tumor recurrence. In particular, by their ability to reinduce cellular proliferation upon reoxygenation, hypoxic HPV-positive cancer cells may serve as a reservoir for tumor regrowth when their oxygen supply is increased. This can occur, for example, through neoangiogenesis , or follHypoxia has the potential to strongly affect the biology of HPV-positive cancers, both through its general effects in tumors as well as through the modulation of the interplay between oncogenic HPVs and their host cell . In the E6/E7 oncogene expression under hypoxia? Secondly, why are the p53 levels not restored in hypoxic HPV-positive cancer cells, although E6 is repressed? Thirdly, how is mTOR signaling maintained in normoxic HPV-positive cancer cells under conditions of E6/E7 silencing and cellular growth inhibition? Fourthly, since the impairment of mTOR signaling is crucial for the ability of hypoxic HPV-positive cancer cells to evade senescence, will it be possible to identify agents which override this regulatory principle and thereby induce senescence in an mTOR-independent manner? Could they be of therapeutic value in combination with RT, CT, RCT, or E6/E7 inhibition, which preferentially act on non-hypoxic cells? Fifthly, in view of the fact that standard cell culture conditions (20% O2) do not reflect the O2 levels in normal cervical tissue (median concentration of 5.5% O2) [Firstly, which molecular mechanisms are responsible for the strong inhibition of 5.5% O2) ,5, are tE6/E7 oncogenes and their host cell thus far have focused on the effects of chronic hypoxia (1% O2 over days) [Moreover, it should be noted that analyses of the crosstalk between the viral er days) . Howeverer days) . Cyclinger days) . The later days) . Enhanceer days) ,77). Inter days) . This fi


A retrospective analysis was performed to describe the cytomorphological and histopathological findings and human papillomavirus (HPV) genotypes for glassy cell carcinoma (GCC) of the uterine cervix. Five cases of cervical GCC, in which the glassy cell features constituted at least 95% of the specimen, were included. Four patients had stage IIB GCCs and one had stage IIIB GCC. All patients underwent concurrent chemoradiation therapy. Based on pretreatment cytology, only 1 of the 5 cases was correctly diagnosed as GCC. The remaining cases were diagnosed as carcinoma of undetermined type, adenocarcinoma, poorly differentiated carcinoma, or unsatisfactory for evaluation. Cytological specimens had moderate cellularity and contained small clusters of tumor cells admixed with amphophilic, granular tumor diathesis. The tumor cells possessed large, round to oval nuclei and abundant, granular, ground-glass cytoplasm. The nuclei exhibited prominent eosinophilic nucleoli. The cytoplasm displayed sharp margins and molding, resulting in “intercellular windows” between neighboring attached cells. HPV genotyping revealed that high-risk HPV types 18, 16, and 31 were detected in 3, 1, and 1 cases, respectively. Consistent with this finding, all cases exhibited block p16 positivity, confirming the association of HPV infection with GCC. In conclusion, a distinct cytoplasmic margin, the characteristic histopathological feature of GCC, was observed in liquid-based cytological preparations. We suggest that sharp cytoplasmic outlines with molding and intercellular windows are characteristic cytomorphological features of GCC. Detection of high-risk HPV in all cases strongly supported the notion that high-risk HPV is involved in the pathogenesis of GCC. Glassy cell carcinoma (GCC) of the uterine cervix is a rare pathological form of cervical carcinoma that occurs in 1-2% of all cases . The WorEarly detection of cervical cancer greatly increases the successful treatment rate. In countries with established early detection programs, the impact of cytology-based cervical screening is reflected in the substantial reduction in the cervical cancer incidence over the past 50 years –27. AlthThe aim of the present study was to describe the cytomorphological and histopathological features and immunophenotypes of cervical GCC. In addition, human papillomavirus (HPV) infection status in cervical GCCs was evaluated. We found characteristic cytomorphological features, and verified that those are consistent with typical histopathological features of GCC. We observed high-risk HPV infection and block p16 positivity in all examined cases.During the period from July 2007 to June 2016, 675 patients were diagnosed as having primary cervical carcinoma. Thirty-three (4.9%) cases of poorly differentiated carcinoma were collected from computerized patient records. Based on the rarity of GCC, we reviewed the 9 (1.3%) cases of cervical poorly differentiated adenosquamous carcinoma. Among the 9 cases, 2 cases that presented glassy cell features but that also had squamous (1 case) or glandular (1 case) differentiation, and 2 cases that had diffuse, strong p40 expression were excluded. The remaining 5 (0.7%) cases were cervical GCCs. Five patients with cervical GCC were included in a retrospective analysis. The median and mean patients ages at diagnosis were 38 and 48.2 years, respectively . Four (80.0%) of the 5 patients had International Federation of Gynecology and Obstetrics (FIGO) stage IIB GCCs and the remaining 1 (20.0%) had FIGO stage IIIB GCC. None of the patients were pregnant or had a recent history of pregnancy. All patients underwent concurrent chemoradiation therapy.The clinicopathological characteristics of the 5 patients with cervical GCC are summarized in Table Histopathologically, the tumor tissue consisted of nests, cords, or sheets of large polygonal cells with a moderate to large amount of pale, evenly spread, finely granular or ground glass-appearing cytoplasm Figure . The tumImmunohistochemical staining results are shown in Table HPV genotype was analyzed using HPV DNA chip assay. High-risk HPV was detected in all cases Table . This reIt has been documented that GCC does not have a distinctive cytological appearance , 3, 17. Previous studies have found a strong association between cervical GCC and high-risk HPV infection , 7–12, wIn the present study, all the five cases we examined expressed block p16 positivity, which was consistent with the presence of high-risk HPV. In addition, focal ER and PgR positivity in 1 case was consistent with a previous study that detected ER (2 cases) and PgR (1 case) among 11 cases of GCC [Cytological differential diagnoses of cervical GCC include atypical reparative cells, nonkeratinizing squamous cell carcinoma with severe inflammation, lymphoepithelioma-like carcinoma, and malignant melanoma. Pak et al.  reportedIn conclusion, we have described the cytomorphology, histopathology, immunophenotype, and HPV genotypes of cervical GCC. We demonstrated that distinct cytoplasmic margins are a major cytomorphological and histopathological feature of GCC. Cytoplasmic molding and intercellular window formation might be useful cytomorphological characteristics for a differential diagnosis of GCC, especially in liquid-based preparations. In addition, high-risk HPV infection in all cases supported the notion that high-risk HPV is involved in the pathogenesis of GCC.Glassy cell features were defined based on the 3 main criteria originally reported by Cherry and Glucksmann  and amplFor liquid-based cytological smears, samples were taken from the fornix, portio, and endocervix. The ThinPrep test  was performed using an automated liquid-based monolayer cell preparation system , according to manufacturer's recommendations. Briefly, the samples were immersed in CytoLyt buffer (Hologic Inc.) and then transferred to a PreservCyt bowl (Hologic Inc.). Cells were released by pushing the brush to the bottom, forcing the bristles apart, and swirling the brush in the fluid. A cylinder containing a filtration membrane was placed in the bowl and rotated to ensure homogeneous cell distribution. Erythrocytes and mucus were allowed to penetrate the filtration membrane under negative pressure, leaving the cell membranes on the filtration membrane. Each ThinPrep slide was fixed in ethanol and stained using the Papanicolaou method. Tumor diathesis, cellular composition, and cellular arrangement of the specimens were evaluated.The biopsied specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. From each formalin-fixed, paraffin-embedded block, 4-μm sections were cut and stained with hematoxylin and eosin. A variable number of hematoxylin and eosin-stained slides from each case were available for review. Among these, the most representative slide, containing an appropriate volume of tumor and possibly normal cervical tissue, was chosen for immunohistochemical staining and HPV genotyping.The formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated using xylene and alcohol. Immunohistochemical staining was performed using the Ventana Benchmark XT automated staining system  or the Dako Omnis , according to the manufacturer's instructions. Antigen retrieval was performed using Cell Conditioning Solution  or EnVision FLEX Target Retrieval Solution, High pH . Sections were incubated with primary antibodies Table . After cWe performed polymerase chain reaction (PCR)-based microarray for HPV genotyping, using a commercially available HPV 9G DNA chip  . The 9G