HPV DNA Array is an E1-targeting PCR genotyping test, with capability of distinguishing 18 high-risk  and 11 low-risk HPV types . HPV DNA Array uses multiplex PCR for E1-gene sequence amplification. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID ELISPOT reader.Aim of the study was to evaluate the clinical performance of the assay against internationally accepted and FDA approved Cobas 4800 HPV test (Roche Diagnostics). Study population comprised of 500 cervical samples.HPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+ detection than cytology (100% vs. 13.6%). The agreement to Cobas 4800 for HPV detection, irrespective of type, was 81.4% with κ = 0.613. The agreement for HPV 16 was 92.8% (κ = 0.929), and for HPV 18 54.2% (κ = 0.681).HPV DNA Array demonstrated good clinical performance for detection of high-grade lesions, and may be considered for usage in a screening setting. Cervical cancer, caused by persistent HPV infection , is an eThere is evidence that HPV-based screening is more sensitive in detecting high grade lesions –10, and The main objective of this study was to report on clinical performance of HPV DNA Array in detection of cervical lesions and to compare its analytical capability to Cobas 4800 HPV test on a study population comprised of 500 cervical samples.HPV DNA Array is a full HPV genotyping assay, newly developed by AID Diagnostika GmbH , which is capable of genotyping separately for 29 HPV types .Five hundred consecutive taken samples of approx. 4000 cervical scrapings collected for the “HEllenic Real life Multicentric cErvical Screening” (HERMES), a study that compared cytology and HPV-based screening  were selFor this study, 2 ml of each sample was used for DNA extraction by QIAamp DNA Mini Kit, in accordance with manufacturer’s instructions. Nucleic acid was eluted in a final volume of 160 μl.HPV DNA Array detects multiplex PCR amplified E1-gene sequences by a reverse hybridization reaction in a 96 well microtiter plate. Oligonucleotide probes, specific for each HPV type, are spotted on a bottom of each well. After hybridization, colored spots were evaluated by ELISpot reader and reading software AiDot . HPV DNA Array is capable of genotyping 18 high-risk  and 11 low-risk HPV types .The person performing the HPV DNA Array testing was blinded to the Cobas HPV results, cytology and histology status of the samples collected.The assays use different target sequences. While Cobas 4800 relies on the more conserved genomic region of L1 the HPV array detects sequences of the E1 that is a larger and more diverse and has more opportunities for type-specific probe sequences and a wider inclusion of genotypes in a multiplexed PCR assay.The Cobas 4800 HPV is a L1-based PCR test with capability of separately genotyping HPV 16 and − 18, and grouping other 12 HR-HPV types  in a single signal.HPV testing was performed as described by Castle et al.  at the LThe cytological smear examination was performed at the corresponding pathology laboratory of the participating hospital where sample was taken. Bethesda 2001 cytology classification guideline was followed . The cytAgreement, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were used as main outcomes. Cohen’s Kappa values determined the agreement between the assays and were interpreted as follows : poor , hence, the HPV DNA Array detected HPV types were grouped accordingly for comparative analytical purposes. Only 14 HPV types covered by both assays were included in the analysis .Statistical analysis was performed with IBM SPSS Statistics for Windows  and MedCalc 15.8 .Four hundred ninety-nine samples were received from the HERMES study from women aged 19 to 66 years, with an average age of 33 years. 217 women were younger than 30 years, 276 were 30 years or older, and for 6 samples, age information was unavailable. Ninety-five samples were HPV negative by Cobas HPV test, and 404 were HPV positive. HPV 16 was detected in 97 samples, HPV 18 in 48 samples, and 321 samples were positive for 12 other HR-HPV types.Cytology result was obtained from 360 women: 274 had normal cytology, 43 had ASCUS, 35 LGSIL and 8 had HGSIL. Biopsy was taken for 74 women: 23 had normal histology, 29 had CIN1, 17 had CIN2 and 5 had CIN3 lesions. No cases of cervical cancer were reported in this population.All 499 samples were tested with HPV DNA Array. An HPV negative result was found in 146 samples, and 353 samples were HPV positive.A discrepancy to Cobas was observed in 90 samples . To exclude test execution mistake, the 90 samples were re-tested with HPV DNA Array, along with 10 concordant samples, as control. All control samples had the same concordant results. In 82 samples, the result stayed the same, still discordant to Cobas, and in 8 samples, the result changed. To confirm which result was true, a third testing was performed, and only HPV types found in 2 or more tests were counted as truly positive. Four samples were initially HPV negative and now became HPV positive, matching the Cobas result; three samples were LR-HPV positive and now showed a co-infection with HR-HPV, and one sample was initially HPV positive for HPV 16, but after re-testing it was twice HPV negative.In summary, after re-evaluation, HPV DNA Array deemed 143 samples to be HPV negative and 356 samples, as HPV positive, among which 25 samples only for types not detected by Cobas, e.g. HPV 42, − 53, − 54, and − 67, hence these samples were additionally placed in the HPV negative group for analysis.We present the analysis on the re-evaluated results.HPV DNA Array showed a sensitivity for detecting CIN2+ lesions of 100% , with a specificity of 9.43% , PPV 31.43% , and NPV 100%  , with a specificity of 0% .A difference between the assays was observed only for detection of low-grade lesions approx. 80% vs. 100% of Cobas, leading to a lower specificity of Cobas test.When compared against cytology Table , HPV DNAHPV DNA Array was positive in 66.3% (331/499) cases, as compared with 81% (404/499) of Cobas HPV test Table .Table 2Hp = 0.000).The agreement between the assays was 81.4%  with κappa 0.613 . The results were stratified according to age; sensitivity, agreement for HPV detection and specificity within < 30 group were 86.2% , 0.618 , 100% , and within ≥30 group 77.4% , 0.593  and 96.87% , respectively. Values of the McNemar’s test deemed the differences statistically significant , hence, the HPV DNA Array results were adjusted accordingly.HPV 16 was detected in 90 samples by HPV DNA Array, as compared with 97 found by Cobas Table , leadingHPV DNA Array detected HPV 18 in 26 samples vs. 48 found by Cobas, demonstrating a sensitivity of 54.2%  with moderate agreement , specificity of 100%  and PPV and NPV of 100%  and 95.3% . McNeimar’s test value was less than 0.05.Twenty-two HPV 18 Cobas positive samples were not detected by HPV DNA Array. Thirteen were single infections by Cobas, and 9 samples were co-infections with HPV 16 and/or other HR-HPV, all of which were detected by HPV DNA Array and only HPV 18 was missed in the multiple infections.Histology was available for 6 samples of 22 missed. We observed that only one sample had a histologically confirmed high-grade lesion (CIN2) and was a Cobas detected multiple infection. In that case, HPV DNA Array failed to detect HPV 18, but other HR-HPV types present in the infection were found.p <  0.05).Sensitivity for detecting 12 other HR-HPV types was 75.4%  with 242 samples detected as compared with 321 by Cobas. Kappa showed good agreement of 0.677 . Specificity, PPV and NPV were 98.88% , 99.18%  and 69.02% . The difference was graded statistically significant , and in one case a high-grade lesion (CIN3) was confirmed. In that case Cobas showed a co-infection of 12 other HR types with HPV 16. HPV DNA Array did detect HPV 16, missing to detect other HR-HPV types.Main aim of this study was to investigate the HPV DNA Array’s ability to detect high-grade lesions by comparing its performance with Cobas HPV test, a validated and FDA approved assay for primary screening, and with cytology, an established method for cervical cancer screening.HPV DNA Array was found to be a simple and short assay . It included a reverse hybridization step, and an ELISA-like staining for assay development. The readout was automated with the ELISPOT reader and an AiDot software that evaluated the full 96 well plate in approx. three minutes. HPV DNA Array is a time efficient assay that allows high throughput, which could be of great benefit for mass screening. Additionally, the HPV DNA Array could be used for epidemiology of HPV, as it fully genotypes and includes most prevalent LR-HPV types (Manuscript in preparation).In this study, HPV DNA Array demonstrated a very high clinical performance with a sensitivity for detecting CIN2+/3+ lesions of 100%, identical to the clinical sensitivity of Cobas HPV test, and higher than cytology (13.6%) in this population. The Cobas HPV test is well investigated and its performance is well documented in the literature. Other researchers reported a similar performance , 20. Howp <  0.05), nonetheless, since all CIN2+ cases were detected, the difference had no clinical meaning.Looking at agreement for HPV detection, irrespective of type, HPV DNA Array showed a good agreement of 81.4% (k = 0.613) as compared with Cobas. This difference was rated statistically significant by McNemar’s test (p <  0.05), and for detection of other 12 HR-types  was found. This is an intrinsic difference due to the different target, primers and probes between the two assay systems compared and the HPV types in the HPV array. It, however, did not reduce the clinical sensitivity of the assay as compared to Cobas 4800. It was observed that although the HPV 18 and/or other HR types were missed, other HPV types present in the infection were detected. Unfortunately, histology information was not available for all of the HPV DNA Array missed cases, however, HPV DNA Array did detect the high risk lesions, therefore, the analytical differences had little clinical bearing. One could suspect that the HPV types missed were not drivers of the infection, but little information is available in the literature.Analyzing the agreement for specific HPV types, a high sensitivity for detection of HPV 16 was observed , which is the most common cancer causing genotype . A lowerThe tendency of HPV DNA Array to have a lower agreement for HPV detection, but a very good agreement for detection of cervical intraepithelial neoplasia to the reference assays, could be explained by the higher number of viral copies in such lesions. Cervical intraepithelial neoplasia tends to have larger viral amounts and is, therefore, easier to detect . It is tWe believe that the performance differences between HPV DNA Array and Cobas could occur due to differences in assay design , genotype spectrum detected, the target HPV gene , and genotyping ability . Evidently, when comparing HPV assays, an ideal high agreement is difficult to reach, as shown by Rebolj et al. . In theiSamples used in this study were received from HERMES study panel for validation purposes and do not represent a screening population. This explains the higher number of HPV positive and lesion positive samples than expected, approx. 80% within this study vs. approx. 10% within screening studies . Within The main limitation of this study was re-testing of the discordant samples with HPV DNA Array and the change of HPV results in 8 out of 90 re-tested samples. This change could occur during PCR and/or hybridization, as a pipetting error, reading error or contamination. Such re-testing was included in the analysis for validation purposes, but would not be feasible during routine diagnostics.This validation study has demonstrated the high potential of HPV DNA Array for detection of CIN2+/3+ lesions. HPV DNA Array has shown a very high clinical sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800; despite the analytical difference in detection of HPV 16 and 18 between the assays. Future studies are warranted to complete the validation and investigate the performance of HPV DNA Array in population-based screening and potentially in low resource settings.


Although suicide risk in veterans has been widely studied, there is little information on the risk of non-fatal self-harm in this population. We used data from the Scottish Veterans Health Study to conduct an epidemiological analysis of self-harm in veterans, in comparison with people who have never served.We conducted a retrospective, 30-year cohort study of 56,205 veterans born 1945–1985, and 172,741 people with no record of military service, and used Cox proportional hazard models to examine the association between veteran status and cumulative risk of non-fatal self-harm, overall and stratified by birth cohort, sex and length of service. We also examined mental and physical comorbidities, and association of suicide with prior self-harm.p < 0.001). The risk was highest in the oldest veterans, and in the early service leavers who failed to complete initial training . The risk reduced with longer service and in the intermediate birth cohorts but has increased again in the youngest cohort.There were 1620 (2.90%) first episodes of self-harm in veterans, compared with 4212 (2.45%) in non-veterans. The difference was statistically significant overall (unadjusted HR 1.27, 95% CI 1.21–1.35, The highest risk of non-fatal self-harm was in veterans with the shortest service, especially those who did not complete training or minimum engagement, and in the oldest birth cohorts, whilst those who had served the longest were at reduced risk. The risk has increased again in the youngest veterans, and further study of this subgroup is indicated. In recent years provision for the health and welfare needs of veterans has assumed increasing importance , and it A number of studies have examined suicide in UK military veterans –7, and oNon-fatal self-harm encompasses both failed suicide and events having no suicidal intent; conversely, suicide may encompass events where a fatal outcome was not intended but the lethality of the method was not appreciated. Intent is rarely documented and, therefore, at a population level, it is necessary to consider all non-fatal episodes together.We used data from the Scottish Veterans Health Study, a retrospective cohort study of 57,000 veterans and 173,000 matched non-veterans, to examine the epidemiology of non-fatal self-harm over a 30-year period in a broad national cohort of veterans in Scotland, irrespective of combat or deployment, in comparison with people having no record of military service. We investigated the association between suicide and a previous episode of self harm. We also examined a range of comorbidities and explored potential risk factors for self-harm so far as was feasible within the available retrospective dataset.The Scottish Veterans Health Study is a retrospective cohort study of all 56,570 military veterans in Scotland who were born between 1945 and 1985, who were resident in Scotland and were registered with the National Health Service (NHS) Scotland both before and after service, and a comparison group of 172,753 individuals with no record of service (‘non-veterans’), matched 3:1 for age, sex and postcode sector of residence . We used Scottish linked health records  and mortality data to compare long-term risk of suicide and major self-harm in veterans and non-veterans, obtaining a single retrospective data download as at 31 December 2012. The study cohort and methods have been described in detail previously [Socio-economic status (SES) was derived from the most recent registered postcode of residence, using the Scottish Index of Multiple Deprivation (SIMD), in quintiles ranging from 1 (most deprived) to 5 (least deprived). SIMD is calculated on a regional basis, in data zones having a mean population of 800, and is based on income, employment, health, education (including skills and training), housing, crime and access to services  to provi‘Non-fatal self-harm’ was defined as the first occurrence of ICD10 code X60-X84, Y87.0, Y10-Y34 or Y87.2, or ICD9 codes E950-E959 or E980-E959, at any position in the record other than the cause of death, excluding any individual whose death was classified as suicide . VeteranCox proportional hazard models were used to examine the association between veteran status and cumulative risk of self-harm, using age as the time-dependent variable, age at first admission for self-harm as the failure time, and age at death (if no self-harm) as the censor time. The use of a time-dependent analysis  protects against immortal time bias , 16. CoxAfter data cleansing to remove incomplete or invalid records, 56,205 (99.3%) veterans and 172,741 (99.9%) non-veterans were included in the analysis. There were 50,970 (90.7%) male veterans and 5,235 (9.3%) female, reflecting the gender balance of the UK armed forces. The mean period of follow-up was 29.3 years, and there was a total of 6.7 million person years of follow-up among veterans and non-veterans combined. The non-veteran group may have included an estimated 327 people who were Reservist veterans (0.2%).p < 0.001  veterans had a record of a first episode of admission to an acute or psychiatric hospital following non-fatal self-harm, compared to 4212 (2.45%) non-veterans. Veterans were at statistically significantly increased risk, unadjusted HR 1.27, 95% CI 1.21–1.35, The increase in risk was only significant for men Table ; for womp < 0.001. Older veterans with between 4 and 9 years’ service also showed an increase in risk but when stratified by birth cohort, the risk was only increased beyond 6 years of service in those born before 1960. The risk was reduced, in comparison with non-veterans, in older veterans with over 16 years’ service, although veterans born from 1960 showed a reduction in risk for all lengths of service beyond 6 years.  birth cohort, comparing age at first episode of self-harm in Early Service Leavers against matched non-veterans, shows that these veterans have fewer pre-service (age 16 and under) episodes but a peak in the early 20s Fig. .p = 0.860. The median elapsed time between the first recorded episode and the fatal episode was 2.0 years for veterans (IQR 0.4–4.3), whilst for non-veterans it was 2.4 years (IQR 0.5–5.8), although the range extended to 13 years for veterans and 23 years for non-veterans. The mean age at suicide for veterans with a previous history of self-harm was 45.5 years (SD 7.9), compared with 43.6 years (SD 8.1) for veterans with no such history, whilst for non-veterans it was 41.6 years (SD 8.6) compared with 41.1 years (SD 9.8). Only 9% of veterans who died as a result of suicide aged under 30 had a previous recorded episode of self-harm, whilst for veterans aged 50 and over at suicide, the figure was 26%. For non-veterans, the change was less marked and the corresponding figures were 16% and 19%.We examined data on 266 veterans and 901 non-veterans who died as a result of suicide. Of these, 50 (18.80%) veterans had a record of a previous episode of self-harm, as did 176 (19.53%) non-veterans. The difference was not statistically significant, p < 0.001. Physical co-morbidities were common; 13% of both veterans and non-veterans who self-harmed had a history of COPD, compared with 4% of the wider veteran and non-veteran population, whilst 7% had a history of peptic ulcer, approximately twice the proportion in the overall population. Diabetes was also more common in those who self-harmed, as were alcoholic liver disease and hepatitis C, although in the case of the latter two conditions, at a lower level in veterans than in non-veterans. Small numbers precluded analysis of multi-morbidity.Fifty per cent of veterans and 46% of non-veterans who were admitted following self-harm also had a recorded secondary care diagnosis of mental health disorder , or psychosis), either concurrently or on a separate occasion, compared with 5% of the wider veteran population and 4.5% of the non-veterans Table . Of thesWe have demonstrated that non-fatal self harm is more common in veterans than in non-veterans matched for age, sex and area of residence, but we have also shown that it is most common in both the oldest and the youngest veterans. The excess risk is restricted to those with the shortest service and is highest in those who left before completing initial training, whilst longer service is protective, suggesting that there is both a ‘healthy worker effect’  and a seAlthough deliberate self-harm and parasuicide (attempted suicide) are frequently considered to lie on the same spectrum, there is good evidence that the two have different underlying psychopathology and should be considered separately, as now recognised by the separate DSM-5 classifications of ‘non-suicidal self-injury’ and ‘suicidal behavior’ , 22. WhiThe literature on gender difference is inconclusive but generally the risk is considered to be higher in women. However a large study in Oxford, UK has provided clarification of any apparent discrepancies by demonstrating a changing ratio with age, with a high female:male ratio for self-harm in adolescents which reverses to give an excess in men beyond the age of 50 . AdverseIn a systematic review of 90 prospective studies of people who had self-harmed, Owens et al. found a median 16% repeating non-fatal self-harm within a year, rising to 20–25% after 4 or more years. The risk of subsequent suicide was much lower, 2% at 1 year rising to 7% after 9 years. The authors suggest that recent self-harm is associated with a 1% risk of suicide, although there is a very low positive predictive value for any method of risk assessment for suicide even in groups of patients considered to be at high risk , 27. In In a study of 1986 male and female military recruits, of whom 4% reported a history of self-harm, self-harming behaviour was strongly associated with negative temperament including aggression, mistrust and manipulativeness, as well as disinhibition and a range of personality disorders including borderline and dependent. Many stated that they self-harmed as a means of relieving tension . The assThe strengths of the present study are similar to those described elsewhere , 12. TheLimitations of the study include possible loss to follow-up of subjects due to migration away from Scotland, which could not be quantified, and the lack of any follow-up data prior to 1981. As the dataset was derived from demographic, vital record and hospital admissions data, there was no information on individual deployments, combat exposure or individual factors such as childhood events or alcohol use. These may represent potential explanatory variables for which we were unable to analyse the impact, or residual confounding for which we were unable to adjust. No information was available about events which did not come to medical attention or were managed solely in primary care; therefore, only the more severe end of the spectrum of self-harm is represented. We have made the assumption that there were no differences between veterans and non-veterans in respect of the setting in which self-harm was managed, other than the frequency of occurrence. Only the last recorded postcode was available which, for veterans, was necessarily post-service. Therefore, the impact of childhood deprivation could not be assessed. Veterans with Reserve service only could not be identified and were included amongst the non-veterans; this could have had the effect of reducing any observed differences between veterans and non-veterans if they also had an increased risk of self-harm, although as the number of Reservist veterans inappropriately classified as non-veterans is likely to have been small, significant impact on our findings is unlikely.Our findings show that the highest risks of non-fatal self-harm are in veterans with the shortest service, including those who did not complete training, and in the oldest and youngest birth cohorts, whilst veterans who had served for longest are at reduced risk. This pattern is not consistent with a causal role for military service but instead suggests that the risk in early service leavers reflects other vulnerabilities such as adverse childhood events. We have also highlighted an important association between mental health disorder, in particular PTSD, and self-harm in ESL veterans. Although overall, fewer than one-fifth of those who died from suicide had a previous record of self-harm, we have illustrated that this risk is higher in older veterans, suggesting that failed suicide attempts in middle age contribute to the increased risk of self-harm which we observed and underlining the importance of treating every self-harm episode in this older age group as a ‘red flag’. The higher risk of self-harm in the most recent birth cohort warrants further investigation. We recommend that specific inquiry into self-harm should be made in all veterans who present with mental health conditions, and especially those with PTSD who left service prematurely.


The majority of cervical cancer (CC) cases are attributable to HPV infection. Altered Notch pathway signals and HPV are believed to modify clinicopathogenesis of CC, however, the involvement of each molecular player and its mechanism is still not known. Jagged-1 (JAG1) is one of the ligands that induce Notch pathway. The involvement of JAG1 in the modulation of a disease condition is not very clear. Hence, this study aims to study the role of JAG1 in HPV-16/18 associated different histological sub-types of CC, especially ADC. 40 non-neoplastic cervical tissues, 30 precancer and 118 tumor specimens  were studied for the expression of the JAG1 protein through immunohistochemistry, immunoblotting and for HPV infection. Two folds increase of cytoplasmic  and nuclear  JAG1 expression was identified in normal (N) vs precancer and three folds cytoplasmic  and nuclear  in N vs. ISCC. Total 85% of ADC patients were found to be infected with HPV, which were 100% infected with HPV-16. These findings suggest the complex synergistic interplay between JAG1 and HPV in regulating clinicopathological progression of CC through its deregulation. Histologically, CC can be characterized by two different sub-categories i.e. (i) invasive squamous cell carcinomas (ISCC) which are frequent and covers 85–90% of CC cases (ii) adenocarcinomas (ADC) which are relatively rare and comprises only 10–15% cases2. The CC develops progression of precancerous lesions; called cervical intraepithelial neoplasia (CIN) grade 1–3 or squamous intraepithelial lesion (SIL)3.Cervical cancer (CC) ranks 45 and more than 90% of detected infections get cleared within two years6. Therefore, the major risk factor of CC is exposure to HPV infection especially HPV-16/18, but to generate and to maintain the malignant processes, HPV infection needs other cofactors; as only few cells infected with carcinogenic HPV develops into CC7. Thus, the current thrust area in the field of CC is to illustrate the molecular mechanism/s involved in the initiation and progression of CC, as well as to develop therapeutic targets.Human Papillomavirus (HPV) has emerged as a fundamental regulator of CC4. The Notch is a complex transmembrane pathway involved in a) cell proliferation, differentiation and development8, b) organogenesis, c) maintains stem cell viability9 and d) renewal in the adult10. There are four Notch homologues (Notch 1–4) and five ligands (three Delta-like and two Jagged/Serrate)11. Among all ligands, animal studies showed that a null mice for Jagged-1 (JAG1) encoded genes exhibit distinct embryonic defects12, suggesting its inimitability independent of canonical Notch pathway. JAG1 binding with Notch-3 dissociates its extracellular unit from the transmembrane unit, sequentially cleaved by proteases, translocating the cleaved intracellular Notch C-terminal fragment to the nucleus and recruits CSL , Mastermind-like proteins  and p300 proteins to transcriptionally regulate target genes.Studies revealed an interaction of HPV proteins with Notch signaling pathwayTill date, there are five studies  increase of cytoplasmic JAG1 expression was identified in N vs. precancer, three folds  in N vs. ISCC. However, only two folds  increase was found in N vs. ADC.JAG1 expression in the nucleus was found to be increased two folds  in N vs. precancer, three folds  in N vs. ISCC and N vs. ADC .In accordance, two folds  increase was observed in N vs. precancer and three folds  elevation in N vs. ISCC. Only two folds  increase was observed in N vs. ADC.JAG1 gene. Moreover, HPV-16 gives an additional signal to the TFs of JAG1 gene  which cumulatively activates the transcription of JAG1 and further results to the increase production of JAG1 protein in the nucleus. The JAG1 protein once formed translocate to the cytoplasm, hence, increasing its level in the cytoplasm.Hence, we hypothesize that JAG1, while binding to Notch-3, gives a signal which results in the cleavage of NICD domain Fig. . The cleReceiver Operating Characteristic (ROC) analysis was done to estimate the JAG1 potential as a diagnostic marker for all three groups  of the uterine cervix.The values for AUC (area-under-the-curve) for JAG1 in precancer , ISCC  and ADC  was found to be significant.Similarly, the significant AUC values for nuclear JAG1 in precancer , ISCC  and ADC  were observed. The sensitivity and specificity for cytoplasmic JAG1 were 76.7%, 84.7%, 75%; and 80%, 80%, 80% respectively. In accordance, for nuclear JAG1 these were 73.3%, 86.7%, 85% and 77.5, 77.5%, 80% respectively {Fig. High sensitivity and specificity of JAG1 in precancer, ISCC and ADC support the clinical utility of JAG1 for early detection and progression of cervical cancer.An association of JAG1 expression with clinicopathological parameters of ISCC has been described as follows Table .Table 2AJAG1 overexpression showed an association with lymph nodes metastasis , vaginal involvement of tumor , as well as with Figo stage  respectively.Tumor vaginal involvement , lymph nodes metastasis  and Figo stage  were found to be associated with JAG1 expression.Similarly, JAG1 was associated with tumor vaginal involvement , lymph nodes metastasis  as well as with Figo stage  respectively.The results imply the association of JAG1 with the aggressive behavior of the CC.A gradual increase in the expression of JAG1 (134 kDa) was identified in precancer, ISCC, and ADC patient samples Fig. , S1a,b. 13. In this study, 85% (17/20) of ADC patients were identified to be infected with HPV. Among them, all the patients were found to be infected with HPV-16 (17/17) and 5.8% (01/17) were found to be co-infected with HPV-18. All the ADC patients had 100% Grade II + III and Figo stage III + IV, and hence all HPV infected ADC were of higher pathological grades.Previously, we identified the prevalence of HPV infection in precancer and ISCC tissuesHence, clinicopathological progression of ADC confirms the involvement of HPV-16.Association of HPV with JAG1 expression in all tissues is defined as follows Table :Table 3CHPV-16 positive precancer patients showed 91.7% (p = 0.0001) cytoplasmic, 87.5% (p = 0.0002) nuclear and 91.7% (p = 0.0007) cytoplasmic + nuclear JAG1 positivity Table . None ofHPV-16 positive ISCC patients showed 94% nuclear and 94% cytoplasmic JAG1 positivity. This showed that in ISCC patients, nuclear (p = 0.0001) and cytoplasmic (p = 0.0001) JAG1 were observed to be correlated significantly with HPV-16.No significant association of JAG1 with HPV-16 positive ADC patients was observed.Increased expression of JAG1 was depicted in Caski (HPV-16 positive) as compared to C-33A (HPV-16 negative) cell lines.This strengthens the hypothesis that HPV-16 associated precancer and cervical tumorigenesis is synergized with altered Notch signaling.Interprotein correlations was analyzed between Notch-3 and JAG1 proteins. In precancer patients, significant positive associations were found between JAG1 nuclear  and Notch-3 nuclear proteins Table . Similar14. The network of altered signaling pathways are also important for the development of CC13. Therefore, for defining an effective treatment strategy, it is essential to understand the molecular mechanism/s involved in CC cells infected with oncogenic HPV types. Knowledge of this may improve the diagnostic modalities for precancer and cancer patients of uterine cervix and defining treatment strategies. This study is the first step towards this direction, where the role of JAG1 in the etio-pathogenesis of HPV infected different histological sub-types of CC is defined. Along with ISCC, we have also included a very rare form of CC i.e. ADC in our study. The outcome of this study will aid clinicians in deciding the targeted therapeutic strategy for CC patients and researchers in understanding the involvement of JAG1 in the pathogenesis of CC.The development and advancement of tumor from precancer to ISCC and ADC of uterine cervix remains a major clinical problem for decades. Evidence suggests that HPV infection alone is inadequate to induce malignant changesJAG1 expression. The JAG1 protein produced in the nucleus accumulates and translocates from nucleus to cytoplasm. Our JAG1 expression results demonstrate altered expression levels of JAG1 protein which results in deregulation of the Notch pathway. This overall may support the infection of the cervical mucosa by HPV-16. Together, this may lead to the acceleration of the cell cycle with an acquisition of more genetic damage. Our results are in agreement with a study15 who showed an intense immunoreactivity of JAG1. However, this study elaborated and showed that cytoplasmic and nuclear expression of JAG1 in HPV associated precancer, ISCC and ADC cases. Based on cytoplasmic and nuclear expression pattern we have explained the probable mechanism of the JAG1 feedback loop.The results of JAG1 protein expression profile through immunohistochemistry suggest the JAG1 binding with Notch-3 triggers Notch signaling in HPV associated CC, which in-turn activates TFs for The ROC curve analysis of JAG1 showed its high sensitivity and specificity suggesting its clinical utility in early detection and progression of CC patients.et al.16 who also identified the correlation of JAG1 expression levels in tumor patients with variable tumor clinicopathological parameters but through western blotting.Immunohistochemical expression of JAG1, with respect to clinicopathological parameters, showed its association with the tumor vaginal involvement, Figo stage and lymph node metastasis, highlighting its clinical utility in ISCC. The upregulated JAG1 expression may interact with aggressive tumor behaviour, tumor progression and expansion, predicting as a potential candidate for biomarker of disease progression. This supports the findings of Yousif et al.16 who identified increased JAG1 levels in CC tissues by real- time PCR and western blotting. This study expanded with respect to HPV associated precancer, ISCC and ADC patients by IHC as well as Immunoblotting.The IHC results corroborated well with immunoblotting and showed concordance with Yousif 13. This indicates that E6 and E7 oncogenic proteins of HPV can interact with the activated JAG1 which in-turn activate various pathways associated in cancers and altogether synergizes with each other, inhibiting apoptosis, promoting cell proliferation and tumorigenesis17. This study did not find any significant association of JAG1 with HPV-16 positive ADC subjects which could be due to the limited number of ADC samples (n = 20) being analyzed.We studied earlier the prevalence of HPV infection in precancer and in invasive CC biopsies, and observed the linkage of HPV positive cancers with clinicopathological parameters of the disease progressionet al.19.We validated the increased expression of JAG1 in Caski, as compared to C-33A cell lines, invasive cervical tumor-derived cell lines. Similar expression pattern of JAG1 was also previously reported in Caski by Veeraraghavalu in vitro cell line experiments strongly support the hypothesis that JAG1 is the major ligand in triggering activation of Notch signaling, which acts as a central player in HPV-16 associated CC development and progression. Therefore, cancer cells expressing JAG1 plays pivotal roles in two manners: (i) JAG1 acts as a ligand and complexed with its receptor activating neighboring cells of a tumor in a juxtacrine way. (ii) The ICD (intracellular domain) of JAG1 may initiate Notch pathway and propagate tumor cell growth, leading to deregulation of JAG1. This creates a favorable environment for the growth of early precancerous lesions, acting as a protagonist in the tumor development and proliferation. Hence, serves a probable transducer of this cancer.Human studies and 21.A better understanding of crosstalk between Notch signaling and other pathways, such as Wnt, Hedgehog, and vascular endothelial growth factor (VEGF), as well as with the immune system can provide valuable clues how to target cancer survival by ablating them and will make JAG1, an attractive approach orchestrated for combination therapy. Furthermore, due to its anti-apoptotic and pro-“stemness” functions, JAG1 blockade epitomizes an alluring model for combination therapy orchestrated by standard chemotherapy as demonstrated in preclinical models of ovarian and pancreatic cancer and lymphomaJAG1 gene due to NICD and HPV-16 signaling to TFs resulting in JAG1 protein in nucleus. However, JAG1 expression in cytoplasm clarifies its translocation from nucleus to cytoplasm which in-turn activates the Notch signaling via binding to Notch-3, hence, acting as a positive feedback loop. Thus, it provides a legitimate target for CC therapy in the hope it might lead to the development of such tailored individualized therapy, as well as characterizing its biological functions in the cells which may be sufficient to abolish the neoplastic phenotype. This may help clinicians in characterizing patients when selecting for treatment of such cancer, by developing chemotherapeutic and combination therapies to interfere with cancer invasion and metastasis. Further validations at an RNA transcript level by RNA Seq can be done in future studies. In addition, studies are desired to assess JAG1 ablation as an adjuvant treatment to existing and currently ineffective chemotherapeutic agents.In conclusion, our findings excavate the understanding of JAG1 driven Notch signaling events in HPV associated progression of CC. JAG1 expression in the nucleus denotes the transcription of 118 tumor specimens , 30 precancer and 40 non-neoplastic cervical tissues  classification of tumor staging23. Each slide was evaluated for its respective histopathological grade and clinical staging by two independent pathologists23. Each collected sample was distributed into two parts, one for histopathological diagnosis and other stored at −70 °C to perform molecular analysis.The clinicopathological characteristics of patients with CC were recorded according to previous studies0C with the primary rabbit polyclonal antibody of JAG-1 (ab7771 Abcam) at a dilution of 1:200. After washing with tris buffer saline (TBS), pH 7.4, sections were incubated consequently with polymer based Envision™  as described by us previously13. The images were captured using Olympus microscope  using Olympus Bio-report software. 3,3-diaminobenzidine hydrochloride (DAB) was used for color development followed by its counter staining by Mayer’s Hematoxylin.All the collected specimens were fixed with 10% formalin, embedded in paraffin and then cut in 5μm sections, further mounted on priorly coated poly-L-lysine  slides. Conventional H&E staining was done in each section followed by immunohistochemistry. Tris EDTA (pH 9.0) was used for antigen retrieval in the microwave and incubated overnight at 413. Percentage positivity score was then combined with intensity scores in order to get the final score.The slides of IHC were independently reviewed by two authors along with one histopathologist independently without prior knowledge of patient’s identity, and were further scored as described previously24. The primary antibodies used were rabbit polyclonal antibody of JAG-1 , and rabbit monoclonal β-actin . Blots were developed using enhanced chemiluminescence ECL detection system . The quantitation and band intensity comparison of JAG1 was done between the subset of normal, precancer and cancer tissues, as well as between C-33A and Caski cell lines respectively by densitometry as mentioned previously24. Alpha Digidoc version 4.1.0  was used for the evaluation of expression.Immunoblotting was performed in representative cases from all categories  of collected biopsy tissues and cell lines according to our previous studieshttp://www.nccs.res.in/) in December 2017 and January 2018 respectively. These cell lines were tested and authenticated directly from NCCS, Pune from where they were purchased. Sixteen short tandem repeat (STR) loci were amplified using commercially available AmpFISTR® Identifier® Plus PCR amplification kit from Applied Biosystems. The cell line samples were processed using the Applied Biosystems® 3500 Genetic Analyser. Data was analyzed using Gene Mapper® ID-X v1.5 software (Applied Biosystems) along with appropriate positive and negative controls. The cells were last tested at the time of their purchase. They were culture in DMEM supplemented with 5% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5% CO2.Human CC cell lines HPV-16 positive (Caski) and HPV-16 negative (C-33A) were purchased from NCCS, Pune  software package was used to perform statistical analysis. Chi-square test was used to determine the expression of the JAG1 protein with the clinicopathological parameters of cancer patients. Interprotein correlations was observed between Notch-3 and JAG1 proteins by Spearman’s rank correlation test. In doing so, the data of Notch-3 was obtained from our previous published reportThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.This study describes the role of Jagged-1 in context to HPV infection, in the pathogenesis of different histological subtypes of cervical carcinoma. It provides the molecular circuitry regulating Notch signaling and a novel therapeutic opportunity of JAG1 ablation, aiding clinicians.Supplement S1, Table-S1, Table-S2,Table-S3, Table-S4, Figure S1, Figure S2


The results indicated that HR-HPV infection is highly prevalent in female coal mine workers in China. Contraception, artificial abortion, working inside the mines and gynecological diseases could increase the risk of HR-HPV infection in these women. HR-HPV DNA load might predict risks of cervical precancerous lesions and cancer. Our findings could provide scientific basis for reducing the risk of HR-HPV infection and cervical cancer in this vulnerable population.High-risk human papillomavirus (HR-HPV) plays an aetiological role in the progression of cervical cancer and precancerous lesions. Determining the risk factors of HR-HPV infection is useful for HR-HPV infection surveillance and control. We aimed to explore the influencing factors of HR-HPV infection in female coal mine workers, and to evaluate the associations between HR-HPV DNA load and cytological and histological changes of cervix. In total 6,325 participants completed standard questionnaire on potential influencing factors of HR-HPV infection and underwent gynecological examinations, HPV test as well as Thinprep cytology test (TCT). 1,512 women with positive results of HPV and/or TCT were referred to colposcopy with biopsy and histological examination. HR-HPV DNA load was evaluated by Digene second generation hybrid capture (HC2) assay. Multiple unconditional logistic regression analysis was used to determine the influencing factors for HR-HPV infection. Of 6,325 study participants, 1,405 (22.2%) were HR-HPV positive. HR-HPV infection rate was higher in women aged 30-50 years, with lower education level, working inside the mines and engaging in shift work. Risk factors for HR-HPV infection in female coal mine workers included contraception , previous artificial abortion , working inside the mines  and history of gynecological diseases , while menopause was a protective factor . The HR-HPV DNA load significantly increased with the severity of cervical cytological ( Cervical cancer is the fourth most common cancer among females worldwide As a coal-rich province in China, Shanxi has a large number of female coal mine workers. Female coal mine workers usually have a relatively lower level of education, younger age of first sexual intercourse, multiple sexual partners, and poor hygiene habits, which have been demonstrated to be risk factors for HPV infection Between September 2011 and October 2012, 6,325 female coal mine workers (age between 20 to 65 years) were enrolled in our study. Subjects were considered eligible for the study according to the following criteria: a) Han people; b) married; c) age ≥20 years old; d) worked in coal mines for at least five years; e) resident in the coal mining area for at least two years; f) not pregnant or lactating; g) no prior history of cervical cancer or precancerous lesions; h) not suffering cancers during the enrollment and has no prior history of tumor of other systems; i) no prior history of treatments for cervix such as Loop Electrosurgical Excision Procedure (LEEP), conization and adnexectomy.All women enrolled in our study were interviewed by trained interviewers using the structured questionnaire regarding their socio-demographic information, working characteristics, reproductive information, history of gynecological diseases and family history of cancers. Then the subjects underwent a gynecological examination and had samples for HR-HPV detection and Thinprep cytology test (TCT) collected. Colposcopy with biopsy and histological examination were given to women with a positive diagnosis of HR-HPV and/or TCT Figure .Informed consents have been signed by all the participants, and this study has been approved by the Institutional Review Board of Shanxi Medical University (No. 2008LL07).DNA load of HR-HPV was evaluated by Digene second generation hybrid capture (HC2) DNA test, an in vitro acid hybridization assay with signal amplification using microplate chemiluminescence for HR-HPV DNA detection. A commercial kit detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 for performing HC2 was used. Alkaline solution was used to denature double-stranded DNA, and the liberated single-strand DNA combined with the RNA probe mix. RNA:DNA hybrids were then transferred to a capture microplate coated with goat polyclonal anti-RNA:DNA hybrid antibodies to be immobilized. Subsequently, alkaline phosphatase-conjugated antibodies to RNA:DNA hybrids were added to react with the bound RNA:DNA hybrids. After washing, chemiluminescent substrate, as the substrate for alkaline phosphatase, was added for signal amplification. Results were analyzed by DIGENE DML 2000 Software. DNA load was expressed by the unit of Relative Light Units/Cutoff Value (RLU/CO), representing the ratio of the light emission of a sample to the average of three positive control samples. HR-HPV DNA load of 1.0 (RLU/CO) or more was defined as HR-HPV positive. HR-HPV viral load was categorized into two groups: low and moderate viral load (<100 RLU/CO) and high viral load (≥100 RLU/CO) Exfoliated cervical cells were collected for cytology test. The cytological evaluation was performed by two cytopathologists at Jincheng General Hospital and the Bethesda Classification system (2001 version) Colposcopic biopsy was performed in subjects with a positive outcome of HR-HPV and/or TCT. Cervix was divided into quadrants and examined each quadrant by gynecological specialists. All visually abnormal areas were biopsied, and the quadrants without a visible lesion were given random biopsies at the squamo-columnar junction. Histological examinations by biopsy were evaluated together by two pathologists. The cases were classified as normal cervix (NC), cervical intraepithelial neoplasia grade 1 (CIN Ⅰ), grade 2 (CIN Ⅱ), grade 3 (CIN Ⅲ) and squamous cell carcinoma (SCC).p values were two-sided, and statistical significance was defined as p<0.05.Influencing factors of HR-HPV infection were analyzed in women with different HR-HPV infection status (HR-HPV-positive and -negative), and associations between HR-HPV DNA load and cervical lesions were analyzed by comparing differences of HR-HPV DNA load in different cervical lesion groups. Count data were examined by Chi-square and trend Chi-square tests. One-way ANOVA was used to analyze differences between groups, and the differences between any two groups were further compared by SNK test. Multivariate unconditional logistic regression was used to determine the influencing factors of HR-HPV infection, and backward stepwise likelihood was used with the inclusion criteria of 0.05 and the exclusion criteria of 0.10. All the data analyses were performed using Statistical Package for the Social Sciences (SPSS) software version 19.0 . All reported χ2trend=35.897, p<0.001). More than half of the women  reported born in Jincheng and the majority  were married. However, HR-HPV infection rate showed no significant association with birthplace and marital status (p>0.05). The socio-demographic characteristics of the study subjects are shown in Table The mean age of our study subjects was 35.31±8.89 years (ranged between 20 and 65 years). Subjects aged between 30 and 50 years old had the highest HPV prevalence (26.5%), and the prevalence declined as age increased. Only 1,850 women (29.2%) received higher education (college/university and above). The HR-HPV infection rate significantly decreased with increasing education level (p<0.05)  were HR-HPV positive. Univariate analysis showed a significant association between HR-HPV infection and workplace, menopause, contraception, contraceptive method, gravidity, parity, abortion history, artificial abortion history, and the history of gynecological disease  (p<0.05). High viral load of HR-HPV was found to be significantly related to cytological  and histological  changes of cervix.In all 6,325 subjects, colposcopy with biopsy and histological examination were given to 1,512 women with a positive diagnosis of HR-HPV and/or TCT. Among them, 946 were diagnosed with NC, 240 with CIN Ⅰ, 157 with CIN Ⅱ, 140 with CIN Ⅲ and 29 with SCC. Associations between HR-HPV DNA load and cytological and histological changes are shown in Table In this study, we found that female coal mine workers had a relatively higher prevalence of HR-HPV with 22.2%, which was higher than population-based prevalence observed in Shanxi Province (12.2%), Shenyang 11.7%), Shenzhen (13.5%) and Beijing (5.8%), suggesting that female coal mine workers might have a higher risk for cervical cancer %, ShenzhIn our study, HR-HPV prevalence in female coal mine workers reached peak at ages 30-50, which might be attributable to the relatively active sex life in women of this age as several studies have shown that HPV could be transmitted to cervix efficiently by vaginal intercourse In our study, significant increase in the risk of HPV infection was observed in women using contraceptives, who have 1.395 higher risk of HR-HPV infection than women who did not, which is similar to a former study Our results showed that artificial abortion was also a risk factor for HR-HPV infection. Artificial abortion can cause damage to the endometrium and mechanical injury to the cervix, so as result in HR-HPV infection. In this study, history of gynecological diseases was also a risk factor for HR-HPV infection. Several studies reported that women with gynecological diseases were prone to persistent HR-HPV infection due to unstable sexual hormone levels, poor function of immune system, lower resistance to HR-HPV and weak ability of viral clearance Results of this study also showed that HR-HPV DNA load and the proportion of high viral load increased with the severity of cervical cytological and histological changes. Huang et al In conclusion, in this study we assessed the influencing factors of HR-HPV infection in female coal mine workers, and found that contraception, artificial abortion, working inside the mines and gynecological diseases could increase the risk of HR-HPV infection. HR-HPV DNA load could predict the risk of cervical precancerous lesions and cancer based on this cross-sectional study. Our findings may provide scientific basis to reduce the risk of HR-HPV infection and cervical cancer in this vulnerable population. Certainly, out of the limitations of the research method we used, the ability to prove the hypothesis of etiology was relatively weak. The prospective cohort studies will be needed to provide more powerful evidence.


Infections with human papillomavirus (HPV) types 16 and 18 account for ~70% of invasive cervical cancers but the degree of protection from naturally acquired anti‐HPV antibodies is uncertain. We examined the risk of HPV infections as defined by HPV DNA detection and cervical abnormalities among women >25 years in the Human Papilloma VIrus Vaccine Immunogenicity ANd Efficacy trial's  control arm.Serum anti‐HPV‐16/18 antibodies were determined at baseline and every 12 months in baseline DNA‐negative women (N = 2687 for HPV‐16 and 2705 for HPV‐18) by enzyme‐linked immunosorbent assay (ELISA) from blood samples. HPV infections were identified by polymerase chain reaction (PCR) every 6‐months, and cervical abnormalities were confirmed by cytology every 12 months. Data were collected over a 7‐year period. The association between the risk of type‐specific infection and cervical abnormalities and serostatus was assessed using Cox proportional hazard models.Risk of newly detected HPV‐16‐associated 6‐month persistent infections (PI)  and atypical squamous cells of undetermined significance (ASC‐US+)  were significantly lower in baseline seropositive vs baseline seronegative women. HPV‐16‐associated incident infections  and 12‐month PI  showed the same trend. A similar trend of lower risk was observed in HPV‐18‐seropositive vs ‐seronegative women .Naturally acquired anti‐HPV‐16 antibodies were associated with a decreased risk of subsequent infection and cervical abnormalities in women >25 years. This possible protection was lower than that previously reported in 15‐ to 25‐year‐old women. A correlation between naturally acquired antibodies to HPV‐16 (and to a lesser extent HPV‐18) and reduced risk of newly detected infection was demonstrated in younger women (15‐25 years) in the control arm of the PApilloma TRIal against Cancer In young Adults .Our aim was to assess whether the risk factors for HPV infection differed between seropositive and seronegative women. We also analyzed risk factors stratified by baseline serostatus to mitigate the limitations in differentiating between new and reactivated infections.22.1Women aged >25 years were included in the control arm of the multinational, VIVIANE trial and were followed up for seven years. VIVIANE is the Human Papilloma Virus: Vaccine Immunogenicity and Efficacy trial. This is a phase 3 double‐blind, controlled vaccine trial based on age, cytology, region, and serostatus.3) and who had sexual intercourse before or during the follow‐up  at Month 0, who had received at least one control vaccine dose (Al[OH]Serum anti‐HPV‐16/18 antibodies were determined by enzyme‐linked immunosorbent assay (ELISA) from blood samples collected at baseline and every 12 months thereafter. Seropositivity was defined as an antibody level greater than or equal to the assay cutoff which was 8 ELISA units (EU)/mL for HPV‐16 and 7 EU/mL for HPV‐18.Liquid‐based cytology samples were tested for HPV using DNA typing PCR‐based assays every six months and cytopathological examinations every12 months.Written informed consent was obtained from each woman before any study‐specific procedures were implemented. The protocol and other materials were approved by a national, regional, or investigational center Independent Ethics Committee or Institutional Review Board. The trial was conducted based on the Code of Ethics of the World Medical Association (Declaration of Helsinki).The endpoints included in these analyses were (a) newly detected HPV‐16 and HPV‐18 incident infections, (b) 6‐ and 12‐month persistent infection (PI), ASC‐US+, and (c) histopathologically confirmed cervical intraepithelial neoplasia grade 1 or greater (CIN1+ and CIN2+). HPV‐16 and HPV‐18 serostatus were the main exposure variables.2.2The analyses were performed on the total vaccinated cohort (TVC) of the control arm of the VIVIANE trial and included all women who received at least one control vaccine dose, who were DNA‐negative for HPV‐16 and HPV‐18 at Month 0, and who also had a normal or low‐grade cytology  at Month 0. All analyses were performed on women who had ever had sexual intercourse before study entry or during the follow‐up period.the type‐specific serostatus at baseline as a binary variable;the type‐specific serostatus as a binary time‐dependent variable;the antibody level as a time‐dependent continuous variable;log‐transformed antibody level as a time‐dependent continuous variable.Analyses were performed using SAS version 9.2. The incidence rate (IR) was calculated as the number of incident events divided by the total person‐time. Person‐years were calculated as the sum of the follow‐up for each participant expressed in years. The follow‐up period started on the day after first vaccination (control vaccine) and ended on the first occurrence of the endpoint or the last visit (whichever occurred first). The relationship between the exposure variables and the risk of newly detected infections or cervical abnormalities was assessed using Cox proportional hazard models. Univariate analyses were done to obtain unadjusted hazard ratios of the determinants of interest (not shown). For each endpoint, the following multivariable Cox models were performed including:Chlamydia trachomatis infection, history of HPV infection/treatment or nonintact cervix. HPV‐associated infection or treatment was defined as two or more abnormal smears in sequence, an abnormal colposcopy or biopsy, or treatment of the cervix after abnormal smear or colposcopy findings. The histories of HPV infection/treatment were collected at baseline using medical history.For each endpoint, we included nine covariates in these models: region, age at inclusion, age at first sexual intercourse, marital status, smoking status at baseline, number of sexual partners during the past year, previous pregnancy, history of For ASC‐US+ only, previous type‐specific HPV infection was included as a time‐dependent variable since the presence of these cells indicates an active infection at a specific point in time. For CIN1+ and CIN2+ endpoints, no inferential analyses were performed due to the low number of cases. Also, analyses of determinants of interest were performed separately for the baseline seronegative and seropositive subjects to help determine whether newly detected infections were new or had been reactivated. The analysis is based on two assumptions: (a) An association between a latent reactivated infection and a known risk factor should be weaker than an association between a new infection and a known risk factor. (b) The reactivation of a PI should be more frequent in the baseline seropositive (representing presumed prior HPV infection exposure) subjects than in the baseline seronegative  subjects.33.1In total, 2687 and 2705 participants were included in the analysis of HPV‐16 and HPV‐18 endpoints, respectively  and 23.3 EU/mL (range: 7‐725) for HPV‐16 and HPV‐18, respectively.C trachomatis‐positive, and 87.8% were classified as pre‐menopausal, 6.5% as peri‐menopausal, 5.1% as post‐menopausal, while the status for the remaining 0.5% was missing.At enrollment, 45% of women were 26‐35 years old, 44% were 36‐45 years old, and 11% were ≥46 years old. Nearly all participants had been previously sexually active at the start of the study, except five who had their first sexual intercourse during the follow‐up. 56% had started sexual activity between 18 and 25 years (32% between 15 and 17), 80% had had one sexual partner during the previous year, and 84% had had a previous pregnancy. Moreover, 14% of women were current smokers, 5% were 3.2The IR per 100 person‐years of newly detected infections was 1.07  for HPV‐16 and 0.64 (0.52‐0.78) for HPV‐18. For 6‐month PI, the IRs were 0.56 (0.44‐0.69) for HPV‐16 and 0.23 (0.16‐0.32) for HPV‐18. For 12‐month PI, these were 0.30 (0.22‐0.40) for HPV‐16 and 0.13 (0.08‐0.20) for HPV‐18.The IRs for ASC‐US+ were 0.34 (0.26‐0.45) for HPV‐16 and 0.21 (0.14‐0.30) for HPV‐18.During the seven years of follow‐up, 13 new HPV‐16 CIN1+ cases, 14 HPV‐18 CIN1+ cases, 8 HPV‐16 CIN2+ cases, and 9 HPV‐18 CIN2+ cases were detected.3.3P = 0.04] and 0.28 , respectively; Table P = 0.26] and 0.53 , respectively). With regard to HPV‐18, we found the risk of newly detected infections and cervical abnormalities was lower in seropositive vs seronegative women, but not statistically significant  showed similar results  were more marked in seronegative than in seropositive women Table .4In this study, HPV‐16‐seropositive women of 25 years and older had a moderate decrease in risk of developing a new type‐specific HPV, PI, and ASC‐US+ compared to seronegative women. This result agrees with the hypothesis that naturally acquired HPV antibodies probably provide only partial protection against subsequent infection with the same HPV type. However, HPV‐18‐seropositive women had deficient levels of protection. Any naturally acquired protection afforded by either antibody is unlikely to be better than the benefits acquired by vaccination. Another study has found that women aged between 30 and 50 who were seropositive for high risk (HR) HPV at baseline had a higher incidence of new type‐specific HPV infection than women who were seronegative.The association between seropositivity and the reduced risk of new infection was less in our study of 26+‐year‐old women than demonstrated in our study of younger women aged 15‐25 years in PATRICIA and in the Costa Rica Vaccine Trial.In the present study, we were not able to determine an accurate antibody threshold value for a defined reduction rate in infection. In the PATRICIA trial, HPV‐16 antibody levels comprised between 200 and 500 EU/mL were associated with a 90% reduction of incident infection, of 6‐month PI and of ASC‐US+.The current study also attempted to consider the change in serostatus during the follow‐up period. Including the serostatus as a time‐dependent variable and as a continuous variable in the Cox models is original. In a recent meta‐analysis, assessing the naturally acquired immunity against HPV infection, none of the 14 included studies considered the possible change of serostatus during the follow‐up period.In another analysis of the control cohort of the VIVIANE trial, the risk of detecting CIN after natural HPV infection in women aged >25 years was similar to that observed in women aged 15‐25 years from the PATRICIA trial.Our analysis of determinants when considered separately for the baseline seronegative and seropositive subjects partially supports the hypothesis suggested by other studies that most of the newly detected HPV infections in seropositive women would be a reactivation of prior HPV infections.The strengths of this study included the large cohort size of approximately 2700 women, and the relatively extended follow‐up period of seven years, which allowed for a thorough evaluation of an unvaccinated cohort. This study also had several limitations. A cervical sample test was performed only every six months, which could have meant that some incident HPV infections were not detected. In addition, it was not possible to determine whether an infection was quiescent, persistent at undetectable levels or was a new infection. Evidence exists that type‐specific HPV infection can present after a period of nondetection.Further research is needed to better understand the natural history of HPV infection and the link between seropositivity and subsequent protection in women of different age groups.In conclusion, multivariable Cox analyses showed evidence of lower risk of newly detected incident and persistent HPV infections and ASC‐US+ in women with naturally acquired antibodies against HPV‐16. The results for HPV‐18 are not conclusive since only a limited and nonsignificant decrease in risk was observed. These findings are consistent with a partial protective role of naturally acquired HPV antibodies against future infection with the corresponding HPV type. However, no threshold of antibody levels necessary for protection could be defined.D Rosillon and F Struyf are employed by the GSK group of companies and received GSK shares. L Baril was employed by the GSK group of companies at the time of the study and received GSK shares. G Dubin is currently a full‐time employee of Takeda Pharmaceuticals, Deerfield, Illinois, and receives salary and stock shares. MR Del Rosario‐Raymundo reports payment of honorarium as principal investigator and support for travel to meetings for the study from the GSK group of companies during the conduct of the study; payment for lectures including service on speakers' bureaus from the GSK group of companies. M Martens reports grants from the GSK group of companies, during the conduct of the study. C Bouchard reports grants from the GSK group of companies, during the conduct of the study. She reports grants and honorarium from Merck. KL Fong reports grant from the GSK group of companies via her institution for the conduct of the study. MC Bozonnat is a consultant outsourced from 4Clinics to the GSK group of companies. A Chatterjee received grant funding for clinical trials, and served on the speakers' bureau and advisory boards for the GSK group of companies and Merck. SM Garland has received advisory board fees and grants from CSL and the GSK group of companies, and lectures fees from Merck, the GSK group of companies, and Sanofi Pasteur. In addition, she received funding through her institution to conduct HPV vaccines studies for MSD and the GSK group of companies. She is a member of the Merck Global Advisory Board as well as the Merck Scientific Advisory Committee for HPV. E Lazcano‐Ponce received fees to conduct HPV vaccines studies from the GSK group of companies and Merck. SA McNeil has received research grants from the GSK group of companies and Sanofi Pasteur and speaker honoraria from Merck. B Romanowski received research grants, travel support, and speaker honoraria from the GSK group of companies. SR Skinner received funds through her institution from the GSK group of companies to cover expenses involved in the collection of data for this study. The GSK group of companies provided funds to reimburse expenses incurred with travel to conference to present data from other studies and paid honoraria to her institution for work conducted in the context of Advisory Board and educational meetings. CM Wheeler's institution received a contract from the GSK group of companies to act as a clinical trial site for this study, and reimbursements for travel related to publication activities and for HPV vaccine studies. Her institution also received funding from Merck to conduct HPV vaccine trials, and from Roche Molecular Systems equipment and reagents for HPV genotyping studies, outside the submitted work. X Castellsagué received research funding through his institution (ICO) from Merck & Co, SPMSD, the GSK group of companies, and Genticel. He also received honoraria for conferences from Vianex and SPMSD. G Minkina, as an investigator at a study clinical site, received fees from the GSK group of companies through her institution. She also received funding from Merck Sharp & Dohme to participate as principal investigator in efficacy trials. She received travel support to attend scientific meetings, honoraria for speaking engagements and participation in advisory board meetings, and consulting fees from the GSK group of companies and Merck Sharp & Dohme. T Stoney received honoraria from the GSK group of companies for study committee membership (Asia Pacific study follow‐up committee for Zoster studies), for conference attendance, and travel support. Her institution also received additional funding from a bioCSL grant for a project in which she is an investigator, funded by National Health and Medical Research Council. She also received travel support for participation in study investigator meetings from Novartis Vaccine and Diagnostics, Sanofi Pasteur, Alios BioPharma, and Pfizer. SC Quek received honoraria and travel expenses from the GSK group of companies for speaking at various symposia. A Savicheva received grants and fees from the GSK group of companies to participate in an epidemiological study (HERACLES). J Salmeron received grants from the GSK group of companies, Qiagen, and Merck Inc. D Money has received grants from Merck, the GSK group of companies, Novartis, and Sanofi for studies conduct. CS Vallejos, TYK Lim, B ter Harmsel, M Cruickshank, A Fiander, and A Ilancheran have nothing to disclose. Click here for additional data file.


Moreover, the differences in cytological alterations in these patients are significant (p < 0.001). The analysis showed a higher prevalence of sexual behavioral characteristics (p < 0.001) and better attention to the execution of the screening test in the Italian population (p < 0.001). Italian women showed more knowledge and propensity to vaccination compared to Turkish women (p < 0.001). Our data highlighted three relevant aspects: the different prevalence of cytological abnormalities, the different distribution of risk factors and, above all, the different attitude of women towards the primary prevention of cervical cancer between an Italian and a Turkish population group.The human papilloma virus (HPV) is a DNA virus associated with benign and malignant lesions of skin and mucous membranes and is the most common sexually transmitted viral infection worldwide. We investigated the prevalence of HPV infection and associated risk factors in Italian and Turkish women population attending the gynecology outpatients clinic in Naples  and Pamukkale (Turkey). Women were enrolled from the Department of Obstetrics and Gynecology of the University of Campania “Luigi Vanvitelli” in Naples  and of “Pamukkale University” in Denizli (Turkey) between January 2014 and June 2015. A questionnaire that included sociodemographic and sexual behavior characteristics, questions about HPV awareness, vaccine status, and reasons for not wanting to get vaccinated, and HPV-related knowledge was completed for each participant, and cervical cytology samples were collected. The prevalence of HPV infection was higher in the Italian group (52.6% vs 32.6%,  Human papilloma virus (HPV) is a double-stranded DNA virus belonging to the Papillomaviridae family and is the most common sexually transmitted viral infection worldwide, and it is associated with the occurrence of condylomas and a variety of cancers in both women and men , 2. The μl for each sample were obtained. One aliquot was used for DNA extraction. DNA extraction from a 500 μl cell pellet aliquot of the cervical sample was carried out using the linear array test , a qualitative in vitro test for the detection of HPV in clinical specimens. The test utilizes amplification of target DNA by polymerase chain reaction and nucleic acid hybridization and detects 37 anogenital HPV-DNA genotypes , 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108) in cervical cells collected in PreservCyt solution. Three experienced cytology experts performed cervical liquid-based cytology tests and formulated cytological reports according to the Bethesda System [t-test. Comparison groups were assessed with Pearson's chi-square test and Fisher's exact test for categorical variables and Student's t-test for continuous variables. A p-value < 0.05 was considered statistically significant. Statistical analysis was made using SPSS for Windows .We performed a retrospective cohort study of outpatient women who came for gynecological examination, between January 2014 and June 2015, to two referral centers: the Department of Obstetrics and Gynecology of the University of Campania “Luigi Vanvitelli” in Naples  and of “Pamukkale University” in Denizli (Turkey). The Local Institutional Review Board approved the study. All participants before enrollment signed a full written consent form. The study was conducted in accordance with the principles of Helsinki Declaration. Inclusion criteria were being 25 years of age or older and active sexual history. Patients with autoimmune diseases or any diseases involving the immune system, with HIV infection, previously vaccinated against HPV, with a presumed or confirmed pregnancy, with a diagnosis of any malignancies or with a history of chemotherapy or radiotherapy, and with a history of total uterine or cervical resection, were excluded from the study. The study was performed according to the STROBE  guidelines . CervicaSeven hundred three women were recruited for the study and 570 participants  were enrolled. One hundred thirty-three patients were excluded according to the exclusion criteria as shown in n=158) vs 32.6% (n=88), p < 0.05. The distribution of genotypes is similar in the groups of positive HPV patients (p=0.325) as shown in Patients were divided into two groups according to the country of the enrollment: 300 patients in the Italian group (group A) and 270 patients in the Turkish group (group B). The comparison between the two groups showed that the prevalence of HPV infection was higher in the Italian group than in the Turkey one: 52.6% . Otherwise, group B showed a greater frequency of atypical squamous cells of undeterminated significance (ASCUS) .p=1.000 and p=0.688, respectively) as shown in Women positive for high-risk HPV were recalled and underwent a colposcopic examination and, if there were an evident lesion, also a targeted biopsy. The results of the biopsies showed a higher incidence of CIN I and CIN II-III in the Italian group and also if not significant (p < 0.05). Other risk factors like smoking, use of oral contraceptives, and sexually transmitted infections were more prevalent in the Italian group but not statistically significant. Moreover, the Italian group showed greater attention to the execution of the pap test as a screening test (p < 0.05), probably related to a higher degree of education (p < 0.05). The distribution of risk factors is shown in The questionnaire revealed a greater prevalence of risk factors associated with HPV infection in the Italian group, in particular for low-age first sexual intercourse and the high number of partners in the last three months (p < 0.05). Moreover, the Turkish group showed a lower willingness to have their children vaccinated against HPV (p < 0.05). Data are shown in The two groups showed an overlapping level of knowledge about the screening for cervix cancer, but, surprisingly, group A showed more knowledge about the vaccine and a higher propensity to use it compared to the Turkish group (HPV is the most common viral infection of the reproductive tract, and most people are infected with HPV shortly after the onset of sexual activity , 22. AltOur data underline three relevant epidemiological aspects that could, if confirmed by larger case studies, address the prevention strategies of the cervical cancer and personalize them on the needs of specific realities: the different prevalence of cytological abnormalities, the different distribution of risk factors among the populations under examination and, above all, the different attitude of women towards the primary prevention of cervical cancer. While in Turkey, it is a priority to create a national database of screening and prevention of HPV that is currently lacking and to launch an information campaign and a vaccination plan. In Italy, instead, the level of knowledge and perception regarding the cervical disease, HPV vaccination, and screening for cervical cancer is still not satisfied, and other studies are necessary to give insight regarding knowledge, perception, and acceptance about the HPV vaccine.


Coincidence of high temperature with terminal reproductive pheno-stages of cotton is chief constraint to achieve yield potential. This high temperature interfere plant defensive system, physiological process, water relations and lint yield production. In this study, we modulated the detrimental outcomes of heat stress on cotton through the foliar spray of nutrients. Cotton crop was exposed to sub-optimal and supra-optimal thermal regimes for a period of one week at squaring, flowering and boll formation stages under glass house and field conditions. Foliar spray of potassium (K-1.5%), zinc (Zn-0.2%) and boron (B-0.1%) were applied at three reproductive stages one day prior to expose high temperature regimes. High temperature increased lipid membrane damage through increased malondialdehyde (MDA) contents in cotton leaves. High temperature stress also reduced leaf chlorophyll contents, net photosynthetic rate, stomatal conductance, water potential, averaged boll weight (g) and seed cotton yield per plant. Various nutrients variably influenced growth and physiology of heat-stressed cotton plants. Zinc outclassed all other nutrients in increasing leaf SOD, CAT, POX, AsA, TPC activity, chlorophyll contents, net photosynthetic rate, stomatal conductance, water potential, boll weight and seed cotton yield per plant. For example, zinc improved seed cotton yield under supra-optimal thermal regime by 17% and under sub-optimal thermal regime by 12% of glasshouse study while 19% under high temperature sowing dates of field study than the water treated plants under the same temperatures. Conclusively, increasing intensities of temperature adversely affected the recorded responses of cotton and exogenous application of Zn efficaciously alleviated heat induced perturbations. Moreover, exogenous nutrients mediated upregulations in physiochemical attributes induced heat tolerance at morphological level. Heat waves along with more number of warm days and nights have been increased in most part of the world2. Cotton crop being native of semiarid regions is highly prone to confront with high temperature at the terminal reproductive stages3. Coincidence of high temperature with reproductive stages of cotton is a chief hindrance to accomplish yield potential in sub-continent. Since, temperature rises to 47 °C in May–June while accompanying high humidity in July–August develops a death-valley for cotton influencing all reproductive stages of cotton crop4. Heat stress mediated impairment in biosynthesis of antioxidants escalates the synthesis of reactive oxygen species (ROS) and thus induces oxidative stress5. Consequently, a cascade of reactions consequence into plethora of ROS that aggravate lipid peroxidation, excessive synthesis of malondialdehyde contents (MDA)6 and ultimately disruption in stability of membranes. Henceforth, the biosynthesis of chlorophyll contents decreases while degradation aggravates under heat triggered oxidative stress7. Whereas, impairment in biosynthesis of chlorophyll ultimately down regulates photosynthesis, translocation of assimilates to reproductive organs and accelerates senescence9. Moreover, heat stress also disrupts stomatal movement and ultimately consequence into poor gaseous exchange. Cotton leaves depict optimum stomatal conductance in temperature ranges 28–30.1 °C10 which starts to decline as temperature rises to 36 °C. Contrarily, as the temperature rises above 40 °C, stomata remain open and photosynthesis is impaired even if soil is well watered. Concurrently, high temperature stress mediated incongruities in stomatal movements disrupts the water relations resulting into reduction of growth12. Perturbations in biochemical attributes ultimately affect morphological attributes. The optimum temperature for development of boll ranges 25.5 °C–29.5 °C. While, boll weight is adversely affected as temperature rises above 25.5 °C–29.5 °C13. Similarly, each 1 °C rise of temperature above day maximum temperature decreases seed cotton yield by 110 kg ha−1 14. Abiotic stresses along with oxidative stress and nutrient deficiency are the major causes of yield reduction throughout world15. Exogenous application of nutrients might prove a potent tool to alleviate deleterious impacts of heat16. Moreover, heat triggered decrease in uptake of nutrients from soil under heat stress further enhance the importance of exogenous supply of nutrients. Moreover, foliar applied nutrients produce higher yield and better-quality produce on alkaline calcareous soils17. Potassium and zinc are immobile within calcareous soils while B availability is also an issue in these soils20. Contrarily, potassium, zinc and boron are required in high quantity during the stress conditions. Potassium, zinc and boron modulates biochemical changes through antioxidant enzymes23 whereas, the exogenous application of potassium, zinc and boron up regulates the biosynthesis of chlorophyll which ultimately delays senescence enhances their quantity; consequently, improves the photosynthetic rate and the photosynthetic enzymes24. Likewise, potassium and zinc mediated regulations in water relations confer heat tolerance by sustaining water and osmotic potential of cell under stress conditions. While, boron availability enhances stomatal opening and thus regulate gaseous exchange under stressed environment26.Temperature is prophesied to rise by 5.8 °C till 2100 and 2.6 °C up to 2050 owing to global warmingTherefore, considering the crucial role of macro (K) and micro nutrients (Zn and B) in protecting crops from extensive range of abiotic stresses, exogenous application under stress conditions might prove a potent tool to alleviate adverse impact of stress. The present study compares the potential role of foliar spray of macronutrient (potassium) in photosynthesis, regulation of water relations and stomatal conductance and micronutrients (zinc and boron) in reproduction and antioxidants. These physiochemical regulations might induce tolerance in morphological attributes of cotton crop exposed to different thermal regimes at squaring, flowering and boll formation. Hence, series of glasshouse and field experiments were conducted with objectives to (1) see the effect of different temperature regimes on leaf physiology and lint yield of cotton and to (2) reveal the role of macro and micro nutrients  for alleviation the impact of high temperature stress.4. Four seeds were sown at 2 cm depth had 12 hours pre-soaking in tap water. At four leaf stage of the seedlings, extra plants were thinned left only one plant in each pot. Treatments were comprised of optimal temperature (32/20 ± 2 °C day/night temperature or no stress), sub-optimal temperature (38/24 °C ± 2 °C or medium intensity heat stress) and supra-optimal temperature (45/30 °C ± 2 °C or high intensity heat stress); exogenously applied nutrients viz. water spray (control), foliar spray of K @ 1.5%, foliar spray of Zn @ 0.2% and foliar spray of B @ 0.1%. One day before shifting the pots to medium and high temperature chambers, the plants were sprayed with either of water (control), potassium (1.5%), zinc (0.2%) and boron (0.1%). Foliar concentrations of these nutrients were optimized in the preliminary glasshouse and field experiments (data not shown). All the plants were grown at 32/20 °C up to 30 DAS (before initiation of squaring). After that, pots were divided into 3 sets, each set was consisted of 20 pots which were transferred to growth chambers maintained for different temperature. First set of 20 pots was exposed to heat stress at squaring, 2nd set at flowering and 3rd set at boll formation. Heat stress was imposed for a period of one week at squaring, flowering and boll formation and data recorded were averaged across the three reproductive stages . Samples from the youngest fully expanded leaves were collected immediately after removing the pots from stress, stored in liquid nitrogen and processed to record various attributes. The experiment was conducted using completely randomized design with split arrangement and replicated four times. Varying temperature regimes were imposed in main pots and nutrients were foliar applied in split pots.The glasshouse experiment was conducted at University of Agriculture Faisalabad (UAF), Pakistan. The experiment was performed during summer 2012. The seed of medium heat tolerant variety (AA-802) was collected from Ali Akbar Enterprises for this study. Soil properties and growth condition were same as have been reported in an earlier studyThe field experiments were conducted at Agronomic Research Area, University of Agriculture Faisalabad, Pakistan during 2012 to 2013. The meteorological data were collected by the Meteorological Observatory of the Department of Agronomy, UAF. Treatments were comprised of sowing dates in main plots viz. early April , early May  and mid-June . While, split plot treatments were consisted of foliar sprays of K, Zn and B viz. water spray, foliar spray of K @ 1.5%, foliar spray of Zn @ 0.2% and foliar spray of B @ 0.1%. Different sowing times were referred as thermal regimes. The varying temperature was recorded at squaring, flowering and boll formation stages. Three sowing times  were selected based on previous five years’ climate data. Sowing of both experiments was done on sandy clay loam soil at times as per treatments during 2012 and 2013. Seed of cotton variety (AA- 802) was collected from Ali Akbar Enterprises during both years of study. Crop was planted with manual dibbling having 75 cm apart ridges and plant to plant distance was 30 cm. Weeds were controlled by two hoeing i.e. at squaring (35 days after planting) and at flowering (60 days after planting) while the sucking insects and boll worms were controlled with insecticides. Nine irrigations were applied as per crop requirement keeping in view the reproductive stages having heat stress at different sowing dates to avoid the drought stress during heat stress periods. The data of physiological parameters were recorded across the three reproductive stages of cotton i.e. squaring, flowering and boll formation. June thermal regime (late sown crop) was considered control, as it provided optimal temperature at all reproductive stages, while April (early sowing) and May sown crops were experienced sub and supra-optimal temperatures at three reproductive stages  at different wavelengths. Superoxide dismutase contents were determined by28 method. Superoxide dismutase was quantified as enzymes units that inhibited photo reduction of nitrobluetetrazolium (NBT) and recorded the absorbance at 470 nm. While, CAT was measured as enzymes units that converted H2O2 to H2O and O2 using the protocol as described by Liu29. The reaction mixture [50 mM phosphate buffer (pH 7) + 5.9 mM H2O2] was mixed with 0.1 mL enzyme extract and read the absorbance at 240 nm. Peroxidase contents were determined using method as given by29. Peroxidase was quantified as units of enzymes that oxidized guaiacol. The reaction mixture was comprised of 50 mM phosphate buffer (pH 5) + 40 mM H2O2 + 20 mM guaiacol and 0.1 mL of enzyme extract per each sample. The absorbance was recorded at wavelength of 470 nm.Leaves samples weighing 0.5 g were extracted in with 10 ml of phosphate buffer (pH 7.8) for the extraction of enzymes. The supernatant was used for enzyme determination after centrifuge and the residues were discarded. The extracted material was stored at 4 °C2O + 100 µL sample extract + 1 mL dichlorophenol-indophenol + 100 µL 0.1% Meta H3PO4 were mixed in a test tube and absorbance was recorded at 520 nm30. Folin-Ciocalteu (FC) reagent method was used for the determination of TPC31. Leaves samples of 0.5 g weight were extracts with 80% acetone (10 mL) and centrifuged. Enzyme extract (20 µL) + FC-reagent (100 µL) + 1.5 mL water were mixed in a cuvette and placed for 30 minutes. Then, added 700 mM Na2CO3 and incubated at room temperature for period of 2 hours. The absorbance was taken at 765 nm having 200 µL sample in each well. MDA contents in cotton leaves were determined according following the procedure as adapted by32. Leaf sample (0.5 g) was homogenized in 10 ml of 0.1% trichloroacetic acid (TCA) solution and centrifuged at 12000 × g for 15 minutes. For each mL of extract 4.5 ml of thiobarbituric acid (0.5%) was used with the reaction mixture and heated at 95 °C for 30 min and cooled. The absorbance was taken at 532 and 600 nm and MDA concentration was determine using formula:−1 cm−1.For the estimation of ascorbic acid, 900 µL dist. H33. The mixture was filtered through a Whatman No 1. A blank with 80% acetone was run; the measurements were taken at 645 and 663 nm through a spectrophotometer. The chlorophyll contents were calculated from the formula:Cotton leaves (0.5 g) were ground in 10 ml of 80% cold acetone and the tubes were stored in dark at 20 °C overnight, indicating minor modifications of previously described methodNet photosynthetic rate and stomatal conductance was determined at three reproductive stages of cotton crop through a portable infrared gas analyzer . The Pn was measured at each reproductive stage after 3 days of spray between 10:00 a.m. to 12:00 p.m. on fully expanded young leaves.34. Leaf water potential was determined through Scholander type pressure chamber  following methodology as described by35 instantly after sampling. While, leaves were stored at −20 °C for a period of one week, then thawed, extracted sap and determined the osmotic potential with the help of osmometer (Osmomat 030).Leaf samples  were collected at pre-dawn (6:00 h) as previously describedTen plants were randomly selected in each experimental unit of filed study while five plants were selected from five random pots of optimal, sub and supra-optimal thermal regimes of glass house study. Averaged boll weight was noted by dividing total seed cotton yield per plant with total number of bolls. While, seed cotton yield was weighed separately for each plot/pot and converted to per hectare yield from each plot.Analysis of variance was employed to determine significance (F-test) of heat and foliar nutrients. While, means of treatments were compared using least significant difference test (p ≤ 0.05). Correlation among the varying response variables was computed using means of treatments calculated across the three blocks. Strength, type and significance of correlation was determined using STATISTIX 8.1 software . Number of pairs of observations (n) to determine correlation were 36 (replications × main plots × sub plots). Figures were developed using MS excel-2016.Significant interaction of heat and foliar nutrients was recorded for all the studied attributes. Supra optimal regime followed by sub optimal regime triggered increase in antioxidants, MDA and decrease in chlorophyll contents, photosynthetic rate, gaseous exchange components, water relations, boll weight and seed cotton yield over the optimal temperature regime. Foliar applied ‘0.2% Zn’ depicted outstanding results regarding the alleviation of adverse impacts of heat, followed by ‘1.5% K’ and ‘0.1% B’ for all the studied attributes Tables .Table 2ESuperoxide dismutase contents were improved by 46% and 25% while catalase contents were increased by 61% and 29% when compared the controls of supra and sub-optimal thermal regimes with the control of optimal thermal regime averaged across of three reproductive stages. Similarly, POX, AsA and TPC contents were increased under sub and supra-optimal thermal regimes. Biosynthesis of SOD was enhanced by 32% and 56% with ‘0.2% Zn’ compared to water spray under sub and supra-optimal thermal regimes. Similarly, ‘0.2% Zn’ instigated improvements in biosynthesis of CAT by 60% and 73% under sub and supra-optimal temperature regime than their respective water treated plants. The SOD and CAT contents were also increased under optimal thermal regime but the effect was more pronounced under sub and supra-optimal thermal regimes. Whereas, POX was up-regulated by 31, 32 and 52% under optimal, sub optimal and supra optimal temperature regimes, respectively with ‘0.2% Zn’ compared to water spray. Likewise, Zn also enhanced the POX, AsA and TPC contents under optimal, sub optimal and supra optimal temperature regimes. Moreover, MDA synthesis was downregulated by 56%, 58% and 57% with ‘0.2% Zn’ compared to ‘water treated plants’ under optimal, sub optimal and supra optimal temperature regimes, respectively (Table a and b contents were reduced by 15% and 66% under the controls (water treated plants) of sub and supra-optimal thermal regimes when compared with water treated plants of optimal thermal regime. Net photosynthetic rate was reduced by 20% and 60% when compared the water treated plants of sub and supra-optimal thermal regimes with water treated plants of optimal thermal regime (averaged across of three reproductive stages). Similarly, stomatal conductance and water potential were reduced while osmotic potential was increased under sub and supra optimal thermal regimes.The Chlorophyll a, b contents, Pn and Gs owing to ‘0.2% Zn’ with respect to water spray were statistically higher under sub and supra optimal thermal regimes. For example, Zn improved Chlorophyll a content by 23% and 46% under sub and supra optimal thermal regimes than water treated plants. Similarly, zinc also improved chlorophyll b contents, Pn, Gs and water potential under sub and supra optimal thermal regimes.  was reduced by 66% and 23% in the controls of supra and sub-optimal thermal regimes than the control of optimal thermal regime. The similar reduction was found for averaged boll weight. The foliar spray of three nutrients  improved SCY by 21%, 16% and 7% in the high temperature regime than water treated plants. Likewise, the nutrients improved the averaged boll weight under high temperature regime Table , Fig. 3.Supra optimal temperature regimes were relatively more detrimental, and it was followed by sub optimal temperature regimes Table . While, In the controls of supra and sub optimal thermal regimes of April and May sown crops, the SOD and CAT contents were increased by 37%, 36% and 22%, 11%  than the water treated plants of optimal thermal regime. Significantly higher activities of SOD, CAT, POX, AsA and TPC with ‘0.2% Zn’ compared to foliar spray of other nutrients were quantified under all temperature regimes for most of cases over the years. However, ‘0.2% Zn’ mediated improvements in biosynthesis of SOD compared to water spray were 32% under sub optimal and 49% under supra optimal temperature regimes averaged across both years of study. Likewise, CAT, POX, AsA and TPC contents were improved by foliar spray of zinc under sub and supra optimal thermal regimes over water treated plant  increased SCY under April and May thermal regimes by 15% and 17% than water spray averaged across during both years of study Table , Fig. 6.The regression analysis under glasshouse conditions indicated that the malondialdehyde has strong negative relation with POX, AsA, TPC and with stomatal conductance. While water relations have positive relation with stomatal conductance Fig. . The rega and b have significant positive relationship with each other (P < 0.05 and P < 0.01) and with CAT, SOD, Pn and SCY (P < 0.05 and P < 0.01). Similarly, Pn and SCY have significantly positive relation with CAT, SOD and with chlorophyll a and b contents (Table a and b have significant positive relationship with each other (P < 0.05 and P < 0.01) and with CAT, SOD, Pn and SCY (P < 0.05 and P < 0.01). Similarly, Pn and SCY have significantly positive relation with CAT, SOD and with chlorophyll a, b contents  affected the membranes of each organelle for example the integrity of chloroplast/photosynthetic machinery37. A balance is required for ROS and antioxidants for the normal functions of plant defensive system. The oxidative stress causes the blockage of nutrients channels38. The high temperature regimes (45/30 °C) of glass house and (April and May) of field study upregulated the superoxide dismutase, catalase, peroxidase, ascorbic acid and total phenolic contents. However, cotton plants were unable to protect the cells from MDA contents due to oxidative stress6 indicating that the optimum temperature for biochemical and metabolic functions ranges between 25 to 31 °C39. In this study, the high temperature stress reduced chlorophyll contents, net photosynthetic rate and stomatal conductance41 indicating that photosynthetic apparatus is most sensitive to heat stress42. It may be the outcome of breakdown of photosynthetic pigments, associated proteins43, reduction in membrane integrity44, inefficiency of PS-II due to disruption of thylakoid structure45 and of linked enzymes46. The reduction in stomatal conductance under high temperature stress47 might be due to the stress signals from roots and consequent production of ethylene48  of field study showed reduction in averaged boll weight and seed cotton yield per plant as reported by14. This may be due to the production of stress hormones52 which may decrease the production and translocation of photo assimilates for developing bolls54.Medium high to high temperature regimes influenced cotton crop physiology and the yieldregimes 4/30 °C of56. This may be due to the reduction in oxidative stress and the stress hormones57.The foliar spray of K, Zn and Boron activated the plant defensive system and reduced the MDA contents from medium to high temperature regimes Tables  and 5 asa and b contents)60  contents, Pn, stomatal conductance and antioxidants showed positive correlation with each other and with SCY while MDA showed strong negative relation with these parameters as reported by6.In this study, foliar spray of potassium, and zinc upsurged the boll weight and seed cotton yield even in K and Zn enriched soilHigh temperature stress at three reproductive stages of cotton crop caused yield reduction which was due to lower boll weight that is associated to less chlorophyll contents and impaired photosynthesis. Exogenous application of macro and micro nutrients  ameliorated the high temperature impact on cotton crop. These nutrients especially K, Zn and followed by B up-regulated the antioxidant enzymes , improved chlorophyll contents, net photosynthetic rate, water relations and seed cotton yield.