Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field. Epigenetic modifications are heritable changes to the chromatin that do not include alterations in the DNA sequence. By regulating the physical structure and accessibility of the DNA, these modifications can switch genes off and on by dictating chromatin conformation either to a transcriptionally active (euchromatin) or silent (heterochromatin) state. Epigenetic marks mainly include DNA methylation and post transcriptional histone-tail modifications. These are obtained through the activity of a range of enzymes and chromatin interacting factors that read, write or erase specific DNA and histone modifications  and histone acetyltransferases (HATs)), histone erasers (histone demethylases and histone deacetylases (HDACs)) and histone readers (like HP1 and MECP2) . WhereasIt is important to note that DNA methylation and post -translational histone modifications appear to work in tandem. Specifically, open or active chromatin is associated with unmethylated DNA and active histone marks whereas closed chromatin is associated with methylated DNA and repressive histone- tail modifications. Together, they constitute the epigenetic memory, which is heritable from mother to daughter cells and in some loci is preserved across generations. However, one of the major differences between these two types of modifications is that DNA methylation promotes stable, long-term repression, while histone post- translational modifications are effortlessly reversible . BecauseEpimutations can be classified on the basis of their origin into primary and secondary epimutations. Primary epimutations are abnormal epigenetic changes that occur without any change in the DNA sequence. One good example is imprinting center defects in rare individuals with PWS . By contBecause secondary epimutations are transmitted through cell generations, they comprise a main target for editing. With the development of editing tools, it has become possible to reverse or overcome epigenetic changes triggered by disease causing mutations. This can be accomplished either through the correction of the underlying mutation, or by rewriting/overcoming the epigenetic marks that are wrongly elicited in the genome. Together with use of programmable DNA binding and nicking platforms, particularly with the CRISPR-Cas9 system, this field of research constitutes a powerful paradigm for deciphering the epigenetic and developmental mechanisms of epigenetic diseases. This short review summarizes and discusses the efforts to date to correct secondary epimutations using a range of editing tools. Since nearly all attempts in human cells to repair secondary epimutations relate to repeat associated pathologies, this review concentrates on the correction of these types of mistakes in repeat associated loci. The application of these strategies for therapy or to other disease associated loci is beyond the scope of this manuscript.FXN) gene in cells from patients with Friedreich ataxia ). Friedreich ataxia (FRDA) is an autosomal-recessive neurodegenerative movement disorder that is caused by insufficient FRATAXIN protein. Most FRDA patients suffer from reduced levels of FRATAXIN due to a GAA tri-nucleotide microsatellite repeat expansion in intron 1 of the FXN gene. When the GAAs expand and reach the pathogenic range (>90 repeats), they incorrectly elicit heterochromatin in the region that surrounds the repeats because of the gain of repressive epigenetic modifications; DNA methylation and histone H3K9me2. This results in an FXN insufficiency due to reduced transcription initiation [To date, only a handful of reports have documented attempts to reverse epimutations by correcting the underlying mutation. Oddly, all experiments have focused on the correction of epimutations that reside in, or act on, repetitive elements. The first study dealt with the deletion of a pathogenic GAA repeat expansion from the frataxin  mutation [FMR1 gene [FMR1, including the promoter sequence [FMR1 gene, reaching mRNA levels that are comparable with those seen in the wild type control. Furthermore, by directing the edited FXS iPSCs to differentiate into mature neurons, persistent expression of FMR1 mRNA and protein levels were achieved even after prolonged differentiation. Overall, this study provided a proof-of-principle that epimutations can be reversed by the repair of the underlying disease-causing mutation. In addition, it implied that the mutation is not only critical for initiating heterochromatin but is also vital for its maintenance. One disadvantage to this study is that the repair was inaccurate and could potentially induce meaningful deletions from the upstream flanking region that may abolish the activity of other regulatory sequences.In a different study CGG repeats were successfully removed from the mutation . FragileMR1 gene . When thsequence . This issequence  applied FMR1 transcription and demethylation in five out of the nine gene edited hybrid clones, while in FXS iPSCs only one out of the five FXS iPSC gene re-activated clones transcription was successfully restored (8 CpG sites). The remaining iPSCs with a CRISPR cut deletion had relatively high methylation levels in the upstream flanking region, implying that epigenetic resetting is not efficient at all times. One explanation for this failure may be the quality of the iPSCs; the cells may have not been fully reprogrammed and retained epigenetic memory which might be more difficult to erase [In a parallel study by Xie et al., the CGG repeat expansion was removed from both, somatic cell hybrid CHO cells containing the human fragile X chromosome and from human XY FXS iPSCs in a more controlled fashion . The expto erase . AlternaC9orf72 locus by gene editing in C9-related Amyotrophic lateral sclerosis and/or frontotemporal degeneration (ALS/FTD). Amyotrophic lateral sclerosis  is characterized by progressive muscle weakness and atrophy due to the degeneration of upper and lower motor neurons in the brain and spinal cord. By contrast, frontotemporal degeneration  affects behavior and cognition, and is caused by the preferential loss of neurons in the frontal and temporal lobe cortices. The leading known cause of ALS-FTD is a GGGGCC repeat expansion in intron 1 of the C9orf72 gene, between noncoding exons 1a and 1b  [C9orf72 [In works that involved extending these studies to other diseased loci, Pribadi and colleagues  attempteutation) ,26. Multutation) , which m[C9orf72 ,30,31 an[C9orf72 ,33. Alth[C9orf72 ,34.C9orf72 alleles in C9/ALS-FTD iPSCs [C9orf72 mRNA levels, as was expected. It would be crucial to examine the effect of demethylation on alternative promoter usage and determine whether it rescues intron 1 retention. This would further substantiate the potential role of C9orf72 hypermethylation in mitigating the toxic effect of RNA/RAN-translation products in C9/ALS-FTD.By targeting the Cas9 to both sides of the GGGGCCs, expansions of 800–1050 repeat copies were precisely excised from heavily methylated TD iPSCs . MethylaDMPK gene [DMPK hypermethylation remains controversial, there is some evidence for an inverse correlation between aberrant methylation levels and transcription levels in the downstream neighboring gene SIX5 [In a different work, Yanovsky-Dagan et al. monitored changes in the epigenetic status of the DM1 locus subsequent to the excision of a large CTG expansion in mutant hESCs and patients’ myoblasts with the myotonic dystrophy type 1 causing mutation . MyotoniMPK gene ,37. WhilMPK gene , it alsoMPK gene . Althougene SIX5  which haene SIX5 ,42. To aWhile most studies have focused on the correction of a heritable mutation that alters the epigenetic landscape of a specific locus (cis elements), there is only one example of the correction of a germline mutation in a trans-acting epigenetic modifier see . In thatFMR1 locus in FXS patient-derived iPSCs with a 500 repeat expansion [FMR1 locus to recruit the demethylating enzyme TET1 by fusion with dCas9. Targeting the repeats efficiently demethylated the entire 5′-end of FMR1, increased H3K27ac and H3K4me3 and decreased H3K9me3 enrichment levels at the FMR1 promoter. As a result, epigenetic FMR1 silencing was abolished, restoring FMRP expression levels in FXS iPSCs and in in vitro differentiated neurons. This procedure had minimal off-target effects in that only 29 out of more than 1000 dCas9-TET1 bound sites presented significant demethylation. While FMR1 expression was increased by 1500-fold, most of the off-target sites showed either none or up to 4-fold changes in mRNA levels. FMR1 expression and demethylation were maintained for at least two weeks after inhibition of dCas9-Tet1 activity and rescued the characteristic electrophysiological abnormalities of FXS neurons. In addition, FMR1 re-activation by epigenetic editing was successful on differentiated neurons, albeit to a lower extent in terms of targeting efficiency and the degree of re-activation. Nevertheless, more than 50% of the demethylated neural precursor cells injected into the mouse brain exhibited FMRP expression after three months, indicating that FMR1 re-activation by demethylation can be preserved for a long period in vivo despite the continuous presence of the mutation. Clearly, this report represents a breakthrough towards realizing the therapeutic potential of epigenetic editing. However, there are several concerns that should be addressed to make this approach feasible. One major difficulty is that it requires the constitutive expression of the epigenetic editor (in this case TET1) in contrast to the direct approach, where the DNA sequence is irreversibly repaired by a hit-and-run targeting method through transient expression of the CRISPR/Cas9 system. Furthermore, unlike DNA methylation in FMR1, the removal of a single modification may not be sufficient to induce the desired chromatin changes that will reactivate/repress the pertinent genes. Other concerns relate to the off-target effects of the system, which depend on the recruitment of the epigenetic editor to the repetitive sequence in FMR1 along with many unrelated CGG repeat elements distributed in the genome, resulting in up to a 4-fold increase in mRNA levels of several unrelated genes. In addition, whereas in FXS cells, multiple copies of CGGs considerably enhance the targeting efficiency, it may not be as effective in other loci which do not possess such long repetitive targetable elements. The alternative for this would be to recruit the epigenetic editor to multiple sites across the locus simultaneously. One major challenge would be to identify the critical elements beforehand that are essential for controlling chromatin structure in that region. Finally, targeting epigenetic editors to specific regions in the genome will not be beneficial if the epigenetic alterations are caused by mutations in trans-acting factors with genome- wide disruptions as in ICF1 (DNMT3B), FSHD2 (SMCHD1), SOTOS (NSD1), RETT (MecP2), and many other epigenetically regulated syndromes.An alternative approach to correcting epimutations that are secondary to disease-causing mutations is to directly target the epigenetic marks rather than repair the DNA sequence . This caxpansion . To do sFMR1 in FXS hESCs with an 800 CGG expansion without altering the DNA sequence. In this case, targeting with a single gRNA directed against the repeats (as opposed to targeting the promoter region) robustly enhanced FMR1 transcription levels in mutant hESCs with a transcriptionally inactive allele despite promoter and CGG methylation, indicating that mRNA transcription is not directly halted by DNA methylation or heterochromatinization. However, in this study, elevated mRNA levels were not sufficient to significantly increase FMRP expression, presumably due to the long CGG tract which impedes translation efficiency [A different approach to epigenetic editing for restoring the normal activity of epigenetically regulated genes is by directly targeting transcription through the expression of dCas9 fused to a transcriptional activator/repressor domain . For exaficiency ,56,57,58ficiency ,60,61.Epimutations may result from heritable changes in the DNA sequence. These defects are the cause of a long list of epigenetically regulated pathologies that result from mis-expression of gene(s) due to inherited mutations that affect the chromatin structure. While in some pathologies the mutation only has a local effect by changing the activity of a cis-regulating element, in others the loss-of-function mutation in a trans-acting factor such as a chromatin modifying enzyme or a chromatin remodeling complex results in a global change in the epigenetic signature of the genome. Very little is known about how and when heritable mutations lead to epigenetic abnormalities. With the advent of recently developed editing tools, it should be possible to address some of these unresolved questions on the mechanism(s), timing and reversibility of epigenetic modifications that are secondary to disease causing mutations . Underst


This paper presents the development and the application of a multisyringe flow injection analysis system for the fluorimetric determination of the major heat-stable known allergen in shrimp, rPen a 1 (tropomyosin). This muscle protein, made up of 284 amino acids, is the main allergen in crustaceans and can be hydrolyzed by microwave in hydrochloric acid medium to produce glutamic acid, the major amino acid in the protein. Glutamic acid can then be quantified specifically by thermal conversion into pyroglutamic acid followed by chemical derivatization of the pyroglutamic acid formed by an analytical protocol based on an OPA-NAC reagent. Pyroglutamic acid can thus be quantified between 1 and 100 µM in less than 15 min with a detection limit of 1.3 µM. The method has been validated by measurements on real samples demonstrating that the response increases with the increase in the tropomyosin content or with the increase in the mass of the shrimp sample. High consumption of seafood has often been associated with health benefits by providing protection and/or symptom-relieving effects for cardiovascular disease , colorecIn addition, seafood can cause food allergy, which today affects 2.5% of the general population around the world ,12. The Several bioanalytical methods for the detection of tropomyosin in shellfish have been developed, such as enzyme-linked immunosorbent assay (ELISA)  or polymo-phthalaldehyde/N-acetyl cysteine (OPA-NAC) reagent after hydrolysis with sodium hypochlorite −) in the negative mode. MSe fragmentation was performed to confirm the structure of the products. MSe experiments were carried out with a trap collision energy ramp from 5 to 20 eV.The ESI source contained two individual orthogonal sprays. One spray was for the column eluent while the other was for the internal standard (lock-mass). During each chromatographic run, leucine enkephalin (2 mg LSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to visualize the total protein content in the extracts, but also as a purification step before LC/MS analysis. Twelve micrograms of protein extract were briefly heated in Laemmli buffer  with ditTropomyosin was excised from the SDS-PAGE gel for mass spectrometric analysis. The SDS-PAGE bands between 34 and 40 kDa  were desSamples such as seafood necessarily require a heavy and complex preparation, but automating detection steps can help save time and money. This study has demonstrated that the detection of food allergens in seafood can be achieved indirectly by an MSFIA system via chemical derivatization by OPA/NAC reagent of a secondary amino acid produced by the acid hydrolysis of tropomyosin, the main allergenic protein in shrimp or crab. The analytical protocol developed showed an increase in fluorescence intensity with increasing mass of the prepared shrimp sample, indicating that the method is robust and without matrix effects.


The emergence of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) and its complications have demonstrated the devastating impact of a new infectious pathogen. The organisational change promulgated by the isolation of affected communities is of extreme importance to achieve effective containment of the contagion and good patient care. The epidemiological study of the population of a small rural community in the North East of Italy revealed how much the virus had circulated during Spring, 2020, and how contagion has evolved after a prolonged lockdown. In the 1st phase, NAAT (Nucleic Acid Amplification Testing) was performed in cases with more or less severe symptoms and a study was performed to trace the infection of family members. Only 0.2% of the population tested positive on NAAT, via nasopharyngeal swab during this 1st phase. In the 2nd phase a random sample of the general population were tested for circulating anti-Sars-Cov-2 immunoglobulins. This showed that approximately 97.9% of the population were negative, while 2.1% (with positive IgG at a distance) of the population had contracted the virus in a mildly symptomatic or asymptomatic form. The main symptom in subjects who developed immunity was fever. Antibodies were found in subjects with forced coexistence with quarantined or infected subjects. The mutual spatial distance by categories has shown higher relative prevalence of IgG positive and IgM negative cases in close proximity but also far from the infected, with respect to an intermediate distance. This suggests that subjects living in thinly populated areas could come in contact with the virus more likely due to intentional/relational proximity, while those living nearby could also be infected through random proximity. The first cases were documented in China (in the Wuhan province) in late 2019 and early 20202. The clinical presentation of CoV-2 infection, named COVID 19, is extremely varied, ranging from completely asymptomatic and / or minor respiratory and gastro-intestinal symptoms to flu-like syndrome, high fever, coughing, shortness of breath, muscle pain, tiredness4 and diarrhea5. SARS associated with high risk of death needing admission into intensive care units occurs only in a minority of cases7. An infected person may develop symptoms after an incubation period that can vary between 2 to 14 days (though up to 29 days has been reported). During this period, they can still be contagious7. Vaccines are currently available and widespreadly used in Europe however, limiting the transmission of the virus and its spread is still important given the possible recurrence of COVID19 waves due to the diminished level of protection over time. Symptomatic or infected subjects must remain in quarantine and contact a doctor immediately to receive the appropriate indications in case of fever persisting above 37.5 with a dry cough (9–3). In March 2020, the World Health Organisation (WHO) proclaimed Europe as the centre of the pandemic. Complete isolation was necessary because of the quick dispersion of the virus and our lack of knowledge about it. But there is now evidence that complete isolations of entire communities may be not necessary to control viral transmission and specific measures can be taken instead10.The 2019 Severe Acute Respiratory Syndrome (SARS) is caused by Coronavirus type 2 (CoV-2)As of November 2021, there are nearly 250 million confirmed cases globally since the pandemic started, with peaks in the Americas, Asia, and Europe. With over 5 million deaths reported, the risk of further escalation of Covid-19 is moderate to high despite Vaccinations and current Sars-Cov-2 knowledge . Recommendations of the WHO on pandemic containment include the widespread use of diagnostic tests, quarantine of cases, contact traceability and social distancing. These are the basic principles of public health and infectious disease control . Because SARS-CoV-2 is a new pathogen, the characteristics of its transmission and diffusion are not yet well known. These include the change of R0 over time and in populations, the heterogeneity of the attack, the percentages of contact between the demographic groups, the interpersonal variation in the communicability and severity of the disease. All are essential to completely understand the spread of SARS-CoV-2 infection within different communities. Differences in population density, cultural and organisational behavior, population age, co-morbidity and contact rates between groups all influence the transmission dynamics within communities. Accordingly, the assumption of a uniform R0 among populations is unrealistic. Furthermore, variation in transmissibility between individuals can play an important role in the spread of SARS-CoV-2. It is unclear to what extent humans are able to generate SARS-CoV-2 immunity.11. Specific antibodies for serum neutralization (NAbs) for SARS-CoV-2 were detected in considerable, although variable, titers in most individuals12, indicating that NAb production against SARS-CoV -2 is relatively common. While these results are promising, other important questions to consider are whether NAb securities will decrease over time and how long acquired immunity will last. Previous studies in confirmed SARS patients have shown that NAb responses against SARS-CoV persisted from several months up to 2 years13. Mass serological testing is therefore needed to estimate how many individuals have been infected, how many are immune and how far we are from reaching the immunity threshold14. The WHO currently recommends that COVID-19 diagnosis be performed by laboratories using SARS-CoV-2 virus-targeted molecular tests. Serological tests are very useful to define the attack rate and immunity in communities which remain a top priority for keeping the pandemic under control15. Rapid antigen tests have now the advantage of rapidity and low costs with high sensitivity and specificity16. Some studies also suggest better accuracy of the antibody tests if performed on processed venous blood compared to capillary blood17. Serological methods will play an important role in the epidemiology of COVID-19 and in determining the immune status of asymptomatic patients, and serology is helpful in combination with nucleic acid amplification tests for the most appropriate diagnosis of COVID-1918. The main tests used for SARS-CoV-2 are performed on nasopharyngeal and oropharyngeal swabs for categories of subjects with symptoms and those potentially exposed19. Nearly all immunocompetent persons develop an immune response following SARS-CoV-2 infection, including B and T cell-mediated immunity due to antiviral humoral and cellular immune responses, respectively. This includes antibodies directed against S and N proteins. Antibodies – including IgM, IgG, and IgA can be detected within 1–3 weeks after infection. IgM and IgG antibodies can arise nearly simultaneously; however, IgM antibodies decay more rapidly than IgG. The observed persistence of antibodies can vary by assay, and some studies have found that approximately 5–10% do not develop detectable IgG antibodies following infection20.Reinfections are rare events and patients who have recovered from COVID-19 have a lower risk of reinfection. Natural immunity to SARS-CoV-2 appears to confer a protective effect for at least a year, which is similar to the protection reported in recent vaccine studies. However, the observation ended before SARS-CoV-2 variants began to spread, and it is less clear how much natural immunity to the wild-type virus will protect against variants21.In the city of Wuhan the prevalence of IgG was 89.8% in COVID-19 patients, 4.0% in healthcare professionals, 4.6% in general workers and 1.0% in other patients. The prevalence of IgG increases significantly with age amongst healthcare professionals and family doctors. The prevalence of IgM antibodies to SARS-CoV-2 was 31.4% in subjects with COVID-19, 1.5% in health workers, 1.3% amongst general workers and 0.2% in other patients22. From 25 May to 15 July, a seroprevalence survey on SARS-CoV-2 was conducted in Italy by the national Statistics Office (Istat) and the Ministry of Health. The Red Cross undertook the operational work. The data show that 1,482,000 people of all encountered the virus, i.e. 2.5% of the population .In Veneto, at the end of the lockdown (from February 22nd to March 9th), there were 744 cases with a daily increase of 70 subjects per day; at the end of phase 1 (from March 10th to May 3rd) there were 18.318 cases and at the end of phase 2 (from May 4th to July 15th) there were 19.220 positive casesAnalysis of symptomatic positive swab subjects during phase 1 of the pandemic.Analysis of IgM and IgG during phase 2 from COVID-19 in a population of about 1000 subjects from a small town during the SARS—Cov—2 pandemic in Veneto . This analysis allows an extrapolated, indirect estimate of the subjects affected by the pandemic.Association between acquired immunity and symptoms, previous quarantine or contacts from previous contagion.Association between acquired immunity subjects and infected subjects.Epidemiological trend within the family and social context.Geospatial distribution patterns of the infection in a small municipality, namely Monastier di Treviso.In order to study the epidemiology of COVID 19 in the population of a small rural municipality in the Veneto Region, the following was undertaken:The lockdown phase from February 22nd to March 9th when the swab was made only on full-blown SARS, i.e. less than 0.5% of the infected, with no serological testing for antibodies;Phase 1 from March 10th to May 3rd, when nasopharingeal swabs were started even in asymptomatic subjects and serological tests began to detect a poor immune response to the virus;Phase 2 from May 4th to July 15th when all suspected COVID 19 individuals began to be swabbed; to monitor all health personnel and guests of nursing homes and hospitals or other health structures; and, especially, young people, even asymptomatic ones. During this phase there was a gradual release of personal and social constrains.During the Coronavirus Pandemic of 2020 in Italy, one can distinguish three different periods:The recruitment for this investigation started on May 25th 2020.The study involved one subject per family within a population of about 4400 people in the town of Monastier di Treviso . This has approximately 1750 families, with an average of 2.49 subjects per family . A total of 922 people was recruited (about a quarter of the area’s population). Recruitment was random and families remained free to participate; generally, the ones most at risk and the ones who had certainly been infected participated. The study was completed within 10 days. The epidemiological situation in Veneto at the end of phase 2 was characterised by Rt of 0.53 . Patients were enrolled after informed consent was provided.Peripheral venous blood samples were collected. Specific qualitative determination of IgG and IgM antibodies directed against the 'new Coronavirus' plasma levels were measured by immunochromatographic method and a two-phase immuno-enzyme sandwich method with final fluorescence detection (ELFA).Medical history information was collected at the time of sampling, namely: 1. presence of symptoms —even in the previous weeks/months; 2. relationship in the family ; 3. previous quarantine or buffer due to risk factors; 4. contact with infected subjects through social and / or work activities.All methods were carried out in accordance with relevant guidelines and regulations, respecting the Privacy of patients .t test for paired data. Simple regression was used for correlation analysis. Groups were compared using the unpaired Student t-test, analysis of variance for repeated measures, or the Fisher exact test, as appropriate. Statistical significance was set at p < 0.05.Categorical data are described by frequency and percentage, continuous data by mean and standard error of the mean. Statistical analysis was performed using the Student Geospatial analysis was performed using QGis v3.14. The sampling density per km2 was calculated using Quartic (biweight) kernel estimator, which reliably mirrors population density. The same approach was used to calculate the spatial densities for individual variables including gender, age groups , most significant symptoms, previous quarantine and contact cases, and IgG positive plus IgM negative cases. All variables densities were represented, both directly and normalised for the sampling density to overcome the masking of effect of the latter being strongly inhomogeneous (i.e. resulting in the percentage of specific cases relative to tested subjects per km2). The centroids of Voronoi polygons were calculated from the dataset, then interpolated using the cubic splines algorithm for visualization of the maps. Nearest neighbour analysis was performed to assess the degree of spatial association between location of immunized subjects and phase 1 infected patients, as follows: The distance matrix was calculated amongst each subject in the study group and the nearest previously infected patient. All study subjects were then grouped into five equally represented classes (by numerosity) of distance; then the prevalence of IgG positive plus IgM negative cases, and the mean age  were calculated for each class.During the pandemic phase of COVID-19 in the Municipality of Monastier di Treviso, 9 positive cases were recorded on nasopharyngeal swab ; presence of symptoms, even in previous months ; previous quarantine; previous contact with infected subjects. This information was not collected due to lack of consent or other issues for 271 subjects (29.4%). It was collected for 652 subjects (70.6%).The characteristics of the study group and the outcome are reported in Table The outcomes of IgG and IgM antibodies determination are reported in Table Analysis of the features of individuals with positive IgG reveals that the average age is 52 years, that under 30 s are only 2 and that those between 30–69 years number 17. There were no over 70 s. Females predominate over males (12/7). It is interesting to note that only fever , quarantined subjects, the contact subjects of certain COVID-19, have a significant correlation in the seeric positivity to IgG. The distribution of patients by age is shown in Fig. Features of IgG positive and IgG negative individuals are represented in Table −2. The density in peripheral areas decreases by a factor of one or more. Females and males are substantially equally represented within the territory, with limited imbalance at the edges , where population density is very low. Age groups are unevenly distributed: young subjects reside mainly close to the urban centre; middle-aged subjects are more homogeneously spread (with limited representation at the extreme edges). older subjects reside mostly in the peripheral, rural areas of the Municipality.As shown in Fig. Based on the patterns of the bulk data reported above, the following most significant cases were selected for geospatial representation: IgG positive and IgM negative; asymptomatic; fever; flu symptoms; previous quarantine and previous contacts.per km2 reported within all the above categories and the overall density of tested subjects , further patterns emerge Fig. .Figure 7Despite the absolute majority of IgG positive and IgM negative cases (11 out of 19) concentrating in the urban centre, peripheral areas (particularly northern) had a significantly higher relative number of cases compared to the urban centre. This is consistent with the relative proportion of subjects with fever or previously quarantined. The relative percentage of patients reporting flu-like symptoms is also concentrated in a restricted peripheral area nearby (N-E), and so is the relative percentage of patients reporting previous contacts with infected patients from phase 1—in a restricted area between the urban centre and the quarantined hot spot. Of particular interest, all the above case categories exhibited a number of involved subjects for inhabitants higher in peripheral rural zones than in the urban centre. This reflects an older age group Fig. . AsymptoNearest neighbour analysis was performed to assess the degree of spatial association between: immunized subjects, age and phase 1 infected patients, the latter being considered as representative of the overall distribution of  infected population during phase 1, rather than as a direct origin of subsequent transmission. Figure 23.The present study describes the prevalence of anti-COVID-19 antibodies in a small municipality in the Province of Treviso, in the Veneto Region, describing the epidemiological trend of Coronavirus during the February-May 2020 pandemic. In particular, screening of at least one subject in each family unit allowed us to estimate how the infection may have spread inside the families of symptomatic and asymptomatic subjects. The low presence of subjects with positive IgG and negative IgM  can be due either to the study phase subsequent to the pandemic peak in the region and the small area of Veneto. It is demonstrated how this virus has had a different circulation than in other areas of the epidemic and how its distribution in Italy is heterogeneous. Rural areas with low population density, as in Monastier have a lower contagion percentage than the areas with higher population density. The lower morbidity in Northwest China might be attributed to its low population density and geographical distance from the epicenter. A previous study proved that high population densities catalyze the spread of COVID-1924.However, vulnerability in certain urban areas can be driven by several factors such as the presence of overcrowded housing and a relatively above-average prevalence of underlying health conditions. This can account for up to 34% variabilityOn the contrary, the serological prevalence observed in Villa Caldari, another small municipality of Abruzzo region was 10.9% which is four times greater than the value (2.5%) reported as preliminary result of a the nation-wide Italian survey on SARS-CoV-2 antibodies. The prevalence observed in the area was also higher than the one estimated for the overall Lombardy region, the most heavily affected region by the pandemic in Italy, which stood at 7.5%. A more similar prevalence was observed in Central Spain and in the area of Madrid (> 10%).25.Although transmission dynamics in a small village like Villa Caldari could be very different from what expected in metropolitan areas, where public transportations and other places with a high concentration of people can play a major role in the spread of the infection. In a small village, other aggregative sites, such as bars, pubs, shops, etc., together with the intense social relationships among relatives, may place a crucial role in the transmission of the infection, as demonstrated by the numerous familial clusters identified, as well as the cluster linked to a bar and tobacco shopSimilarly in our rural area, the subjects who lived with infected people developed immunity in 25% of cases. This indicates how forced coexistence, but respecting hygienic-sanitary and behavioural rules of "isolation within isolation", can reduce infection, when compared to those who had not come to know with certainty that they were living with an infected person. Only 9 subjects with a positive buffer were found in the Municipality of Monastier di Treviso during the Pandemic Phase, with an incidence of 0.2% of the total population. Forced cohabitation in the family environment during the lockdown contributed to the spread of the infection within families, while the isolation of those infected in nursing homes for the elderly protected other residents from the risk of infection Fig. .Moreover, from the geospatial distribution of the homes of the subjects with positive IgG in the Municipality of Monastier, it is evident that the housing proximity of families affected by Covid is very relevant from an epidemiological point of view (distribution may be defined as a "leopard’s spots"). In fact, subjects with positive IgG lived in or near areas inhabited by positive subjects, as determined by nasopharyngeal swab.From the epidemiological trend it is possible to deduce from the graph of the prevalence of positive outcomes. These vary as a function of the average distance of the subjects from the nearest positive patient (result positive on NAAT). As prevalence was very low, we cannot identify statistically significant trends. However, in qualitative terms, there is an interesting bimodal trend, with higher prevalence for those who live very close (less than about 200 m) or far (more than about 1.3 km) from a positive buffer zone. In fact, these two factors could represent two different transmission modes: those who live very close to infected people (more likely in urban centres) are more likely to come in contact with the virus due to random proximity effects. Those who live far from infected people (more likely in the suburbs or countryside) have a higher probability to come in contact with the virus due to intentional / relational proximity. Among other things, the calculation of the average age between the distance classes confirms the impression from the maps where the more advanced (more sensitive) age groups tend to be relatively more localised in less densely populated areas. This, combined with the above interpretation, substantially confirms the obvious fact that those who are older usually live in the suburbs (and probably move even less). Also, they encounter transmission more by relational contact than by casual proximity.25.In the Spanish cohort there was a higher parental incidence, rather than being linked to a stable family relationship compared to single individuals (0.7%) or grandparents. Living in a context of isolation (even forced) has allowed grandparents to maintain immunological integrity, due to the absence of infection. The subjects who did not remain in quarantine due to the absence of contact with those infected, or did not report contact with the infected, but in forced lockdown, demonstrated a reduced possibility of contagion. By contrast, the subjects in quarantine, having been in contact with an infected person were themselves infected and developed immunity in 50% of cases, even if asymptomaticThe distribution by age allowed us to understand how the middle age (from 30 to 69 years) was more affected, given the greater frequency of movements of subjects, often for work job reasons, as compared to elderly subjects (over 70), who remained in isolation from the rest of the family. Even in young subjects (under 30) the circulation of the virus was low, probably due to lockdown rules being widely respected. Also, there are fewer younger people in this—mainly rural—municipality. In asymptomatic subjects , only 3 were positive. This demonstrates that if the virus has a low circulation in an area, asymptomatic subjects are less dangerous and lockdown prevents contagion. Their lower mean age, which is a generally associated with better health, could have further reduced risk of severe infection.25. A study from Henan Province of China showed that an asymptomatic carrier transmitted COVID-19 virus to her five family members20; therefore, the management of asymptomatic patients is critical for preventing outbreaks. Seventeen percent of patients were asymptomatic carriers, all of whom were detected during the period of quarantine or medical observation23.Approximately one-third of seropositive participants were asymptomatic in the Spanish-based study similary to this study25.The symptom that was mostly correlated with remote antibody COVID-19 positivity was fever (20% in febrile subjects). Indeed, in 163 symptomatic subjects  hyperpyrexia was the most frequent symptom (24.5%). Of these, only 4.9% were IgG positive. On the contrary, subjects with general flu symptoms (27% of the symptomatic ones) antibody positivity to COVID-19 was as low as 0.6%. In the Spanish study by Cito et. al amongst the symptomatic infections, the most frequent symptoms are consistent with COVID-19. In our population, dyspnoea was poorly reported, possibly because outpatients not presenting severe respiratory distress composed it. Anosmia (the loss of the ability to detect one or more smells) should be recognized as an early symptom of COVID-19Limitations of this study are related to the difficult diagnostic approach at the beginning of Sars-Cov-2 Pandemia.SARS-CoV-2 has caused a global pandemic and is associated with significant morbidity and mortality. Serological tests appear to be unreliable during the first infectious period. However, IgM may play a role in subjects with at least 14 days after the onset of symptoms attributable to Covid-19. The presence of IgG antibodies within a population is useful to understand how widely the virus has circulated in the weeks and months following the epidemic. The heterogenic epidemiological distribution of Sars-Cov2 infection in different rural and metropolitan areas (a "leopard’s spots" distribution) has been demonstrated by different studies. Aside from anamnesis of fever, quarantine and isolation with infected subjects which have been linked to IgG positivity, subjects who live very close to infected people are more likely to come into contact with the virus due to random proximity effects. By contrast, those who live far from infected inhabitants have a greater probability of coming into contact with the virus by intentional / relational proximity, determining the concept of reciprocal spatial distance by categories.


To transfer sterile medical devices (SMD), infusion bags (IB), ampoules (A), injection vials (V) and infusion bottles (B) into a laminar airflow cabinet (LAF) or safety cabinet (SC) with a surface bioburden as low as possible.Surface bioburden of the outer layer of SMD, IB, A, V and B was determined by contact plates. Surface bioburden determination of critical spots on A, V and B (ampoule necks and stoppers) was determined by high-recovery swabs and contact plates. Particle emission from white cardboard boxes was determined by a particle counter.The chances of a contaminated outer layer of SMD is negligible as long as they stay in their original boxes. The outer layer of double-packed IB can contain a considerable number of micro-organisms. As found in previous studies, the surface bioburden of A, V and B is low as long as they stay in their original cardboard boxes. Particle emission from white boxes is low. The necessity of a final disinfection step inside LAF/SC of critical sspots of A, V and B cannot be proven. SmallSMD, ampoules and injection vials can be transferred into the background areain their original white boxes. Other materials have to be unpacked in front ofthe lock while the operator wear disposable gloves. Disinfection of the outerlayer of IB, before transfer trough the lock, is advised. Tohave materials with a low chance of contamination in LAF/SC, transfer bypresentation for SMD and IB and using a sterile tray for disinfected materialsis an effective procedure. Wiping of ampoule necks and stoppers inside LAF/SC isadvised based on risk assessment.Small SMD, ampoules and injection vials can be transferred into the background areain their original white boxes. Other materials have to be unpacked in front ofthe lock while the operator wear disposable gloves. Disinfection of the outerlayer of IB, before transfer trough the lock, is advised.When SMD, ampoules, injection vials and infusion bottles stay in their original boxes as long as possible, the aseptic transfer and the disinfection procedure can be maintained effectively and efficiently. During aseptic handling many materials are used. These can be divided into materials with a sterile surface and materials with a non-sterile surface. Materials with a sterile surface are sterile medical devices (SMD) and double-packed infusion bags (IB). Materials with a non-sterile surface are glass and plastic ampoules, and injection vials and infusion bottles, all usually packed in cardboard boxes.Materials are transferred in two steps into the working area (laminar airflow cabinet (LAF), safety cabinet (SC) or isolator (I)). The first step is the transfer through a lock from an adjacent area into the background area , plastic needles (Codan filter straws) and syringes  were taken aseptically from the location in the background area where they were stored. Samples were transferred into LAF or SC and the paper surfaces were monitored by contact plates . Contact time was 10 s. Steel needles were examined in sets of five and plastic needles and syringes were examined separately. For the number of samples, see 2 of the whole surface was examined.To get the paper surface as flat as possible, fingers were held on the back of this surface see . To impr10.1136/ejhpharm-2019-002034.supp1Supplementary dataAfter sampling, the contact plates were incubated for 7 days at 30+/-1°C and cfu were counted after 3 and 7 days.In the background area of different hospital pharmacies, samples were taken aseptically out of their original boxes:In three hospital pharmacies, 10 samples of 100 mL NaCl 0.9% IB In four hospital pharmacies, 10 samples of 500 mL NaCl 0.9% IB In five hospital pharmacies, 10 samples of 2000 mL parenteral nutrition (PN) bags 2 of the whole surface was examined.The surface bioburdens were determined by contact plates . Contact time was 10 s. To improve contact, the plates were turned a little from left to right several times with light pressure. Only one 55 mm-diameter contact plate was used for each sample, which meant that up to a maximum of 23.7 cmAfter sampling, the contact plates were incubated for 7 days at 30+/-1°C and cfu were counted after 3 and 7 days.In the background area in 10 hospital pharmacies, 10 samples of four different kinds of ampoules and vials were taken aseptically, just before disinfection. The sampled products were: 10 mL plastic ampoules (10 x Addamel Kabi Fresenius), 10 mL glass ampoules , 10 mL injection vials  and 100 mL infusion bottles .Samples were transferred into LAF or SC and monitored by contact plates as described in part A.After sampling, the contact plates were incubated for 7 days at 30+/-1°C and cfu were counted after 3 and after 7 days.3 air per minute). The distance between the probe of the particle counters and the rubbed boxes was 10 cm.The experiments were executed in a SC. The original white cardboard boxes with Soluvit N, Vitintra adult 10 mL and Supleven 10 mL , and empty white carboard boxes used in the pharmacy for packaging ampoules and vials, were rubbed together continuously. Particles were counted five times during 4 min with a Met One HHPC 2+handheld airborne particle counter  and injection vials  were taken straight from their original boxes and placed in a LAF cabinet. Plastic flip-off caps were removed from the vials. Two sampling methods were used:Swab: 20 ampoule necks and 20 vial stoppers from non-disinfected and disinfected* glass ampoules were thoroughly wiped by a moistened high- recovery nylon-flocked swab (Quantiswabs bioMerieux) and directly streaked on a TSA plate .Contact plate: 35 non-disinfected and 35 disinfected* vial tops  were pressed with light pressure and with a holding time of 10 s on TSA plates .*Disinfection according to the one-step two-towel disinfection method as described in part B.After sampling, the TSA plates were incubated for 7 days at 30+/-1°C and cfu were counted after 3 and after 7 days.2 of the outer layer of wrapped SMD. To be able to assess the distribution of cfu over the different samples, SD are given.2 of the outer layer of double-packed IB. Because of the great variety in cfu counts on the 119 IB (between 0 and more than 50 cfu per investigated surface), the results are subdivided into different groups . High cfu counts were not correlated to a particular manufacturer, kind of bag or volume.Hospitals 3 and 5 had the overall best results  and Hospital 2 had the worst results .3. The maximum limits for airborne particles in Grade C in operation are also presented in The mean number of particles from each kind of cardboard box after five-times rubbing are expressed in The results of the bioburden determination on the vial stoppers and the ampoule necks are summarised in For designing an effective and efficient transfer process we needed information about the surface bioburden of different kinds of materials with and without a sterile surface, stored in different places. A lot of information is already available in parts A and B.2 at a maximum). It should not be interpreted as information on the whole surface bioburden but can be helpful for the redesign of the transfer process.The results in Because of the low number of samples and the differences in the transfer processes in the participating hospital pharmacies, the results in The risk of contamination via the airborne route is low because aseptic handling is done using closed systems. Different studies have confirmed this.As mentioned in the Introduction, the chance of a contaminated outer layer of SMD will be negligible as long as they stay in their original boxes. After opening, contamination will occur as shown in Infusion bags are also sterilised in a second outer layer (see Introduction). Compared with the surface bioburdens of non-disinfected ampoules and vials , the outAs mentioned previously, it is important to have materials with a low-surface bioburden before disinfection.In Hospital pharmacies 3, 4 and 5 ampoules and injection vials are stored in their original white boxes up to use. The surface bioburdens are low and are comparable with the bioburdens in materials stored under the same conditions, as described earlier.Cardboard, however, is not recommended in the background area because of particle release. Our experiments on particle emission contradict this recommendation see . From thTheoretically, particle emission from white laminated cardboard should be lower than from white non-laminated cardboard. Our results, however, did not confirm this expectation see .Cardboard is a well-known source of viable particles, among which are spore-forming bacteria.Like ampoules and vials, the surface bioburden of infusion bottles is low as long as they stay in their original boxes.The optimal transfer for materials with a sterile and a non-sterile surface into the background area is summarised in Only a part of the material has to be unpacked before transfer.Disinfection can be restricted to IB (and infusion bottles in the case of harmonisation).Storage of ampoules and vials inside the background environment is easier to handle in boxes compared with single items.Starting point for the procedure described below is working with two operators.Before use, SMD have to be unwrapped. If unwrapped materials are brought into LAF/SC the risk of contact between critical spots  and the disinfected, but not sterile, surface of the worktop is relatively high. Therefore, we advise that the second operator partly unwraps SMD in front of LAF/SC. This operator presents the sterile side to the first operator see  who pull10.1136/ejhpharm-2019-002034.supp2Supplementary dataAs explained earlier, we advise keeping disinfected materials in a sterile tray on a sterile surface  are used.The transfer of these materials into a laminar airflow cabinet, safety cabinet or isolator is a critical process from a microbiological point of view.Ampoules, injection vials and infusion bottles have a low-surface bioburden as long as they stay in their original boxes.The chance of contaminated outer layers of sterile medical devices is negligible, as long as they stay in their original boxes.When sterile medical devices, ampoules, injection vials and infusion bottles stay in their original boxes as long as possible, the aseptic transfer procedure and the disinfection procedure can be maintained effectively and efficiently.


Limited evidence is available on apparent temperature (AT) and hospital admissions for acute cardiac events. We examined the associations of AT with admissions for acute cardiac events and acute coronary syndrome (ACS), and explored the effect difference between ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction ACS (NSTE-ACS). Poisson regression with distributed lag non-linear model was applied to examine the temperature-lag-admission associations. Stratified analyses were performed by gender and age-groups for acute cardiac events. A total of 11,657 acute cardiac events admissions were collected from hospital-based chest pain centers in Beijing, during 2017–2019. The single day effect of low AT  appeared on the 2nd day and persisted until the 11th day, with estimated relative risk (RR) ranging from 1.44  to 1.084  for acute cardiac events and from 1.034  to 1.006  for ACS. The single day effect of high AT  was only observed on the current day. The cold effect on acute cardiac events was more pronounced among female and older patients. The cumulative effect of high AT on STEMI admissions and low AT on NSTE-ACS reached a peak RR peak of 2.545  and 3.71  on lag 0–6 days, respectively. Both high and low ATs were associated with increased risk of acute cardiac events and ACS admissions. STEMI admissions may be more sensitive to high AT while NSTE-ACS to low AT. In China, IHD ranked the second leading cause of death and approximately 1.7 million deaths were attributable to IHD in 20162. Acute coronary syndrome (ACS) represents an acute manifestation of IHD with a high risk of death, encompassing unstable angina (UA), non-ST-elevation myocardial infarction (NSTEMI), and ST-elevation myocardial infarction (STEMI). According to the latest guidelines, UA and NSTEMI were categorized as non-ST-elevation acute coronary syndromes (NSTE-ACS) because of the similarity in clinical manifestation and management strategies4. Given the vital importance of immediate medical care for patients with urgent or emergent causes of ACS, it is necessary to identify some modifiable risk factors and take precautionary measures to avoid treatment delays as much as possible, which may help reduce the mortality.Ranking the first cause of disability adjusted life years (DALYs) in 50-years-and-older population in 2019, ischemic heart disease (IHD) has been one of the ten most important drivers of increasing disease burden over the past three decades worldwide11. Extreme temperatures were also linked to increased daily number of hospital admissions, emergency department visits and ambulance dispatches16. The chest pain is one of the most common chief complaint among the emergency department patients and is associated with potential life-threatening acute cardiac events. Although a number of studies have reported the association between temperature and morbidity of AMI , few studies investigated the association stratified by subtypes of AMI. Besides, previous studies indicated an important role for temperature in the incidence of ACS17. However, epidemiological studies evaluating the effect of temperature on incidence of ACS are relatively scarce, let alone on effect differences between STEMI and NSTE-ACS22. In view of a threefold increase in the rate of hospitalization for NSTEMI whereas a small reduction for STEMI, improving awareness of the preventive and medical care for NSTE-ACS is important23.Meteorological factors including ambient temperature, humidity, sunshine, atmospheric pressure, have been reported to be associated with mortality or morbidity of cardiovascular events such as myocardial infarction, out-of-hospital cardiac arrest25. As a combination of meteorological indicators, including ambient temperature, relative humidity and wind velocity, apparent temperature (AT) has been proven to reflect the thermal sensations experienced by the human body more objectively than ambient temperature alone26. AT has been adopted to represent the combined exposure of temperature and humidity in several studies exploring the impact of temperature on mental health, behavior and some other diseases29. However, the study using AT to quantify relationship between temperature and onset of acute cardiac events has not yet been reported.In addition, some studies proposed that raw temperature indicator may not accurately describe the true thermal effects, because human perception of ambient temperature depends on both temperature and other meteorological factors such as humidity and wind speedThe aim of our study was to explore the impacts of AT on daily hospital admissions due to acute cardiac events in Beijing, China, from 2017 and 2019. Furthermore, we sought to explore whether the AT-admissions associations varied by gender, age and types of ACS.30.Beijing is a typical northern city of China and is located at latitude 39°56′N and longitude 116°20′E, with a permanent population of 21.54 million in 2018 . Beijing has a monsoon influenced humid continental climate with hot humid summers and cold windy dry winters. The annual average temperature ranges from 10 to 12 °C, with average daily minimum temperature of − 6 °C and average daily maximum temperature of 33 °Chttp://data.chinacpc.org/). All of the ten hospitals passed chest pain center accreditation in 2017  and are designated hospitals for patients with an acute cardiac event in Beijing. Therefore, the data collected from China Chest Pain Center Data Reporting Platform is enough to represent patients with an acute cardiac event during the study period in Beijing. This data reporting platform served as a national surveillance system to evaluate the characteristics, treatments, and outcomes of patients hospitalized with acute cardiac events, launched by the Chinese Cardiovascular Association. To be eligible, we included data from those who meet the following criteria: (1) are aged 18 years or older; (2) are admitted by all kinds of modes including directly by self, via emergency medical services, transferred-in or in-hospital; (3) have a primary diagnosis of cardiogenic chest pain, including the STEMI, NSTEMI, UA, aortic dissection, pulmonary embolism, and others.Data on admissions due to acute cardiac events in 10 hospital-based chest pain centers in Beijing from March 1st, 2017 to February 28th, 2019 were obtained from the China Chest Pain Center Data Reporting Platform (http://data.cma.cn/). Air pollution data including carbon monoxide (CO), nitrogen dioxide (NO2), particulate matter with aerodynamic diameter less than 10 µm (PM10), particulate matter with aerodynamic diameter less than 2.5 µm (PM2.5), sulfur dioxide (SO2) and ozone (O3) was collected from Beijing Municipal Ecological and Environmental Monitoring Center (http://www.bjmemc.com.cn/). We averaged the daily mean concentrations of air pollutants from the available monitoring data across seven national air monitoring stations evenly distributed in urban districts of Beijing. Extreme low and high daily temperatures are defined as those daily mean temperatures that fall below the 2.5th percentile and above the 97.5th percentile of the total daily apparent temperature distribution, respectively32. Using this percentile-based definition, the extreme low temperature threshold is less than or equal to − 11 °C, and the high extreme threshold is greater than or equal to 34 °C for the period studied.We derived meteorological data from the China Meteorological Data Sharing Service System , water vapor pressure (hPa) and average wind velocity (m/s), respectively. The water vapor pressure e is calculated from Eq.  on daily hospital admissions.34. The model included the following confounding factors: day of week (DOW), public holidays (Holiday), PM10 and O3. The lagged and potential non-linear effect of AT were evaluated by distributed lag non-linear model (DLNM)35 by adding a cross-basis function with up to 14 lag days, using a quadratic B-spline with three internal knots at the 10th, 75th, and 90th percentiles location of AT distributions and three knots in log response36.A generalized linear model (GLM) approach with a quasi-likelihood Poisson distribution for dispersion data was conducted to investigate the association between daily hospital admissions and extreme temperature conditions (both low and high). A natural cubic B-spline with seven degrees of freedom per year was used to control for the long-term trend and seasonal cycles32. MAAT represents the minimum of the exposure–response or the temperature at the lowest risk obtained from specific model. Thus, the cold and heat effects were defined as the hospital admission risks at the 2.5th and 97.5th temperature percentiles, compared with the minimum-hospital admission temperature percentile. The approach is well documented38 and has been used to estimate the impact of temperature on mortality. Stratified analyses were performed by gender and age groups for acute cardiac events admissions. And the analyses for different diagnostic types of diseases  were also conducted through the above steps. The RR for each category was compared to the reference MAAT and reported with both 95% confidence intervals (CI). All statistical analyses were carried out in R software (version 3.6.3) using “dlnm” package38.A new metric Minimum Admission Apparent Temperature (MAAT) at minimum relative risk was defined in this study, which is similar to Minimum Mortality Temperature (MMT) defined by previous studiesEthical approval for the study was obtained from the institutional review boards of the GUSU group ethics committee (GUSU19005), established by Chinese Cardiovascular Association. The need for informed consent was waived by the Institutional Review Board because the health information was primarily the hospital specific daily count of admissions, i.e., summarized data  without any individual identifiers. The study was performed in accordance with the Declaration of Helsinki.We included a total of 14,447 chest pain admissions  in 10 public hospitals in Beijing between March, 1st 2017 to February, 28th 2019 (730 days). Among these acute cardiac events admissions, male and patients aged above 65 years accounted for 65.1% and 42.3%, respectively , such that the risk of acute cardiac events onset is higher at both extreme high and low ATs relative to the MAAT. The effect of low AT on acute cardiac event admissions appeared one day after the exposure and can still be observed on the 8th and 11th days after the exposure whereas the effect of high AT was immediate and relatively short-lived. In addition, the current study found that MAAT of STEMI is close to extreme low temperatures, indicating increased risks at high temperature. Conversely, the minimum risk temperature of NSTE-ACS is close to extreme high temperatures, indicating that extreme low AT tend to have significant effect on NSTE-ACS rather than high AT.Overall, our findings contribute to the limited epidemiological evidence for the adverse effects of non-optimum extreme temperatures on the onset of acute cardiac events and incidence of ACS in the area of subtropical monsoon climate, and may be of major population and clinical implications. We adopted the new comprehensive index of AT as a potential warning index, which may be a more realistic and objective measure than ambient temperature alone. The associations of extreme ATs with acute cardiac events admissions may offer implications regarding mobilization and improved utilization of public and emergency resources in the city. For example, in high- or low-temperature weather conditions, public service announcement or ads and banners could be used to alert the public. Similarly, emergency medical system and emergency medical staff could also be alerted for a possible surge in calls and services. Adaptation strategies such as using air conditioners in summer and adequate hydration should be considered. Although the risk of climate and environmental triggers to any single individual is relatively small compared with the effect of well-documented risk factors (e.g. smoking), the public health relevance is considerable, and small percentage increases can translate into substantial absolute increases, because the incidence of chest pain are common and exposure to temperature can be ubiquitous.14. Our study showed that the TMAAT for all-cause chest pain (23 °C) and acute cardiac events (22 °C) admissions were close to each other. We found significant increased risks for both at extreme low and high ATs, and the RRs for acute cardiac events were larger than those for all-cause chest pain. In addition, the heat effect for acute cardiac events could be observed on the current day while it could not be observed until on the 6th day after exposure for all-cause chest pain. This inconsistency in research findings could be due to the major difference in dataset used, study population, analytical method as well as temporal and spatial properties.Our findings are consistent with several studies reporting the impact of extreme temperature on admissions due to ACS or chest pain. A study exploring impact of extreme temperatures on ambulance dispatches in London reported that the minimum ambulance dispatch temperature (TMADT) for chest pain was less than the 1st temperature percentile and found increased risks only at higher temperature. Whereas the TMADT for acute cardiac events was above the 99th percentile indicating increased risks only at higher temperatures40. Besides, the ethnicity of the study participants, the climatic conditions of study area and the environmental background may contribute to the differences in the results41. These discrepant findings indicate that further large-scale epidemiological studies and subsequent meta-analyses are thus needed in different climate zones to determine these relationships with certainty.Our results did not find cumulative effects of extreme ATs on acute cardiac events admissions though they showed large RR values for cold effect on the second day after exposure and on the present day for heat effect. This might be inconsistent with what past studies found with their data. One possible explanation is that widespread use of central heating system in Beijing, a typical northern city in China, have improved the adaptation to cold ambient temperature in winter and usage of air conditioner in summer could relieve the hot stress in summer44. A possible explanation for this increased susceptibility in the elderly relates to a reduced thermoregulation, together with a decreased capacity in perception of changes in body temperature45.In addition, the present study observed larger size of cold effect estimates in those over 65 years old. Several previous studies reported similar findings that extremely low temperature was more likely to induce an acute cardiovascular event in susceptible individuals such as the elderly6. However, a research from Japan found that both lower mean temperature and increased maximum temperature from the previous day were independently associated with the STEMI occurrence throughout the year46. With regard to the difference between the impact of temperature on STEMI and NSTEMI, a study comparing the seasonal variation of STEMI and NSTEMI in Israel demonstrated significant increases in STEMI cases during the winter months whereas no statistically significant differences in the seasonal variation were found for NSTEMI47. Furthermore, seasonal variations in the incidence of ACS could reflect differences in the underlying pathobiology48. Although our results are not the same, these findings do support the concept that the underlying pathophysiology of STEMI is different than that of remaining types of ACS. And inconsistency in these findings merits further investigation focusing especially on the comparative investigation of differences in relationship between temperature and subtypes of ACS. Also, further larger scale original studies or systematic reviews concerning the causal effect of short-term exposure to extreme temperature on subtypes of ACS risk are needed to resolve this discrepancy.A novelty of this study is the comparative investigation on the relationship between AT and specific subtypes of time-sensitive acute cardiac events and ACS (STEMI and NSTEACS) whilst most previous studies have examined emergency department visits in general or a single disease . There is no agreed conclusion about how effects of temperature may vary on different subtypes of ACS. In the present study, admissions due to STEMI was only affected by extreme high AT while NSTE-ACS admission was found to be only affected by extreme low AT. Contrary to the association of AT and STEMI in the present study, a study conducted in two large Italian urban areas reported that colder days were the days of higher risk of STEMI28 and identified as the main predictor of heat-related mortality49. To date, however, the relationship between AT and admissions due to acute cardiac events or ACS has not been reported in literature. Compared with previous studies using daily mean temperature as exposure variable, AT can provide a measure of the combined effect of environmental variables on human health and wellbeing, and can estimate the human perception of temperature50.Another novelty in the present study is that we employ a biometeorological index AT as exposure measurement to evaluate the effects of temperature on acute cardiac events, rather than daily mean, minimum or maximum temperature. Human body is exposed to a set of atmospheric parameters simultaneously and AT could capture the reaction of the human body to the combined conditions of the thermal environment. It has been used in some studies exploring the impact of ambient temperature on mental and behavioral health51. However, our analysis failed to control for influenza as a possible confounder due to the unavailability of data, and the results may show an overestimate of the effect in cold seasons.There are several potential limitations of this study. First, this is a retrospective ecological study and the ecologic fallacy cannot be ruled out. While causation cannot be directly inferred from our work, ATs were all individually capable of predicting risk well before the adverse event. Second, the single-center and relatively small sample size of this study may limit the generalization of these findings to the whole population. And thus, the association of AT with acute cardiac events and whether the effect varies by subtypes of ACS should be explored in future studies. Third, meteorological data were extracted from fixed monitoring sites rather than individual exposure measures, which may lead to measurement errors. However, we linked the daily emergency admissions of each hospital to the daily meteorological measures in the corresponding district to minimize the misclassification. Fourth, seasonal influenza was associated with an increase in MI-associated deaths and hospitalizations in previous studiesBoth high and low extreme ATs were found to be risk factors for hospital admissions due to acute cardiac events and ACS. The study also observed detrimental effect of high extreme AT on STEMI and low extreme AT on NSTE-ACS. These findings may help better understand the relationship between temperature and the onset of acute cardiac events, and will contribute to developing public health intervention measures, as well as planning of clinical service and emergency resources to reduce mortality caused by untimely emergency treatment in ACS patients. Further multi-center studies aiming to differentiate the effect of non-optimal temperature on ACS subtypes are warranted.Supplementary Information.


The main characteristics of cervical cancer are abnormal and uncontrolled cell proliferation, and it regulates cell growth, differentiation, and cell death through genetic and epigenetic changes. This paper mainly discusses the radiosensitivity of the cervical cancer protein kinase B signaling pathway and discusses the specific mechanisms that affect the occurrence and development of cervical cancer. In addition, this paper studies the effect of transient transfection knocking down the expression of TRIP4 in cervical cancer cells on the expression of key proteins in related signaling pathways and explores the mechanism of its specific effects and finds the mechanism of TRIP4's effect on cervical cancer radiosensitivity. The findings of this study show for the first time that knocking down TRIP4 inhibits cell viability by inhibiting the P13K/AKT and MAPK/ERK pathways, and this corresponds to the first part of the experimental results, which show that knocking down TRIP4 inhibits colony formation and increases apoptosis in HeLa and SiHa cells. Moreover, simultaneous inhibition of TRIP4 and hTERT proteins can increase the radiosensitivity of cervical cancer cells. These findings indicate that the inhibition of TRIP4 may be a new type of treatment that selectively targets the P13K/AKT and MAPK/ERK pathways and hTERT pathways in cervical cancer cells and provides a therapeutic option for the treatment of cervical cancer. Cervical cancer is the fourth most common cancer among women worldwide, and most cases occur in developing countries. Moreover, cervical cancer accounts for nearly 12% of female cancers . BecauseP = 0.004), and the ORR of patients treated with bevacizumab was also higher . However, 8% of patients in the experimental group reported genitourinary fistulas and gastrointestinal fistulas, which brought new challenges to these previously irradiated patients [In the treatment strategy of cervical cancer, simple cytotoxic treatment has little effect. Therefore, in recent years, a new method for the treatment of cervical cancer has been proposed, that is, targeted drug therapy, whose purpose is to inhibit specific molecules involved in key cell pathways. At present, the most promising method is to use targeted therapy in combination with one or more traditional chemotherapeutic drugs. Bevacizumab is a recombinant humanised monoclonal antibody that targets a circulating vascular endothelial growth factor. It was authorised by the US FDA in 2014 as the first targeted medication for cervical cancer therapy (VEGF). For patients treated with bevacizumab, regardless of whether it was accompanied by combined radiotherapy and chemotherapy, OS was improved by 3.7 months  and mediates the interaction of transcription activators or related ligands. Recent studies have shown that ASC-1 can play a role in the development of tumors and act as a coactivator of the transcription factors AP1, SRF, and NF-KB in HeLa cells. At the same time, studies have shown that ASC-1 is located in the nucleus and cytoplasm of rat fibroblasts and regulates the expression of antiapoptotic molecule plasminogen activator inhibitor-2 (PAl2) in gastric cancer cells under different conditions. A recent report showed that ASC-1 promotes the progression of melanoma by regulating the expression of COX-2/iNOS. However, as a subunit of ASC-1, the role and mechanism of TRIP4 in cervical cancer are not fully understood. The impact of TRIP4 on cervical cancer can be used as a new discussion.This article studies the radiosensitivity of the protein kinase B signaling pathway in cervical cancer to provide a theoretical reference for the treatment of cervical cancer.Aneuploidy in human tumour cells includes aberrant chromosomal shape and number, resulting in chromosome instability. Mitosis is one cause of instability, and mitotic mistakes during chromosomal separation may result in chromosome loss and excess .Protein kinases have been found in eukaryotic cells from the beginning of time, and they have remained relatively unchanged throughout their evolution. Furthermore, researchers have investigated the whole protein family in a number of model animals, as well as its function in other species. At this time, mammalian cells have been shown to express three protein kinases: protein kinase A, protein kinase B, and protein kinase C . ProteinInitially, researchers found that protein kinase A and protein kinase B were overexpressed in primary breast cancer and colon tumors. Later, researchers found that protein kinase A was amplified in these subgroups of tumors, and this amplification was associated with poor prognosis for breast and colon cancer patients. Protein kinase A expansion or overexpression has also been discovered in various cancers, such as breast cancer, pancreatic cancer, ovarian cancer, and gastric cancer. Protein kinase A, protein kinase B, and protein kinase C mRNA levels were measured in several primary tumour samples from various stages and origins in a recent study . Protein kinases A and B were both significantly overexpressed in these tumour samples when compared to the control of normal tissues. Surprisingly, the expression levels of protein kinases A and B appear to decrease and increase in parallel, while protein kinase C does not appear to be overexpressed, and its expression levels have no correlation with protein kinases A and B. The close correlation between the expression of protein kinases A and B in tumors is very interesting and suggests that there is a feedback mechanism between these two proteins .Abnormal protein kinase A can cause mitotic errors, resulting in the production of aneuploidy or deviation of chromosome copy number, and ultimately lead to cell death or induce malignant transformation. It is speculated that the possible mechanisms of protein kinase A involved in transforming cells are as follows: (1) the high expression of protein kinase A leads to centrosome amplification and aneuploidy. In this case, some oncogenes may be activated and the function of tumor suppressor genes will be lost. (2) The abnormal activity of protein kinase A may participate in the development of tumors by regulating the p53 pathway. (3) It may affect the balance between apoptosis and division. (4) Protein kinase A can participate in transforming cells by inducing telomerase activity. However, overexpression of protein kinase A does not cause all cell lines to become malignant, and overexpression of protein kinase A may be accompanied by amplification or mutation of other associated genes, such as p53. Numerous studies have shown that there is a clear correlation between protein kinase A and malignant tumor aneuploidy. Therefore, the abnormality of protein kinase A may be related to the pathological characteristics, clinical stage, and prognosis of malignant tumors. However, the results of studies on different tumors are different. The literature  studied Cervical cancer is characterised by aberrant and uncontrolled cell proliferation, which is regulated by genetic and epigenetic alterations and controls cell development, differentiation, and cell death. Cervical cancer is the leading cause of mortality from gynaecological cancer in developing nations like China, with HR-HPV infection accounting for 80-90 percent of cases. They have a role in integrating viral DNA into chromosomal DNA to activate cell proto-oncogenes and oncogenes, as well as in the silencing of tumour suppressor genes. Cervical intraepithelial neoplasia (CIN) is caused by these functions, which speed up cell growth. The main genetic mutations and clinical diagnostic characteristics may be linked to variations in the development of illnesses with comparable clinical and pathological symptoms. As a result, biomarker discovery utilising genome and proteome methods is expected to allow individualised treatment and open up new pathways for disease diagnosis, classification, and treatment .Phosphoinositide 3-kinase P13K)/AKT and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) oncogenic signaling pathways have often attracted attention in cervical cancer research. The reason is that they are often overactivated in cancer, which causes the loss of control of cell metabolism and apoptosis and enhanced cell survival and proliferation . CompareK/AKT and5 cells/well). After 48 hours, the cells were transfected with a control vector LacZ or a plasmid overexpressing TRIP4. TRIP4 targeting ShRNA was used to silence its expression in HeLa cells. Four specific shRNA plasmids and a control nontargeting shRNA plasmid fused with mCherry were purchased from GeneCopoeia. Virus packaging and transfection were performed according to the Lenti-Pac TM HIV Expression Packaging Kit User Manual (GeneCopoeia). Finally, cell lines SH1 and SH2 with higher TRIP4 silencing efficiency were selected by IF and Western blot analysis.TRIP4 plasmids with various length gene sequences were created in the lab and utilised in transfection studies. The Lipofectamine 3000 reagent is used in this article for transfection. In order to exaggerate TRIP4 in HeLa or SiHa cells, the cells were seeded in a 6-well plate . The slides were immersed in lysis buffer  for 1 hour and washed with neutralization buffer for 5 minutes, 3 times each time. Then, the slides were placed in cold electrophoresis solution (300 mM NaOH and l mM EDTA) and electrophoresed at 300 mA setting 25 V for 25 minutes. The cells on the slide were then stained with ethidium bromide solution (20 μg/ml), and images were captured using a fluorescence microscope. The number of cells with or without a comet tail was counted and averaged, and the experiment was repeated 3 times [After irradiating the grouped cervical cancer cells with 6 Gy, the collected cervical cancer cells were resuspended, and then, they were spread on agarose-coated glass slides  were mixed and incubated on a rotary shaker at room temperature for 2 hours during incubation. Then, the DNA-protein complex was pulled down by centrifugal precipitation. The protein-complex was washed with PBS and analyzed. The cells were grouped and cultured in 6-well plates. When the cells had grown too large, we fixed them with 1% formaldehyde, scraped them off the plate with a cell scraper, collected and cleaned them by centrifugation, and then sonicated them on ice to cleave the DNA into 200 bp to 500 bp pieces. As a DNA input control, one-third of the total cell lysate was utilised. Anti-TRIP4 or nonspecific IgG immunoprecipitated the remaining two-thirds of the lysate. The immunoprecipitated DNA was subjected to PCR to amplify the hTERT promoter fragment. The PCR products were electrophoresed on a 2% agarose gel, and the ethidium bromide staining was observed under a microscope. Each experiment was repeated 3 times under the same conditions, and statistical analysis was performed using SPSS 19.0 and GraphPad Prism 5.0. The results are shown as the mean ± standard deviation (SD), and the t-test is used to compare the values of the test and the control. P value < 0.05 indicates that the result is statistically significant .The synthetic biotin-labeled double-stranded DNA probe corresponds to the hTERT promoter sequence from nucleotides -902 to +40 bp. Then, 500 P < 0.05). The above Western blot results indicate that P13K/AKT may participate in the TRIP4-mediated cervical cancer growth promotion in the MAPK/ERK signaling pathway . After that, we further explored the degree of DNA damage in the four groups of cells after irradiation. The comet experiment showed that after irradiation, the comet tail of the TRIP4 knockdown group was longer than that of the control group (P < 0.05), suggesting that DNA damage increased and the ability of cells to repair DNA was weakened. After that, Western blot immunoblotting was used to identify changes in the expression of DNA damage repair proteins (Rad51 and p-H2AX proteins) in the four groups of cells from the two cell lines. As shown in P < 0.05).In the case of transient transfection knocking down the TRIP4 protein of the HeLa cell line and SiHa cell line, in order to verify the effect of TRIP4 on cell DNA damage repair ability, we divided the two cell lines into untreated group Mock, simple irradiation group Mock+IR, TRIP knockdown group 1+IR, and TRIP knockdown group 2+IR. Each batch of cells was passaged onto 6 cm culture plates and grown at plate clone density. The radiation dosages were 0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy, and each group was repeated three times for each dose once the cells had established a colony. After culturing the cells for 48 hours, a radiation survival curve was made based on the number of colonies. The results showed that the survival rate of the TRIP4 knockdown group is lower than that of the control group , and theol group  (P < 0.0P < 0.05), indicating that TRIP4 and hTERT proteins may have a direct or indirect regulatory relationship. The specific mechanism should be verified through further experiments.As mentioned above, there is a close relationship between telomerase and radiosensitivity, and radiation especially increases telomerase activity in cancer cells. In order to determine whether TRIP4 inhibits the radiosensitivity of cervical cancer cells by affecting telomerase activity, we transfected HeLa and SiHa cell lines with TRIP4-SiRNA and simultaneously detected the changes in hTERT expression when TRIP4 was knocked down. When the TRIP4 protein of the knockdown HeLa cell line and SiHa cell line is transiently transfected, the expression of hTERT protein is inhibited. However, when the TRIP4 protein of the HeLa cell line was overexpressed, the expression of the hTERT protein increased. Therefore, there may be a regulatory relationship between the two. Through quantitative analysis of Western blot bands, it was found that the hTERT expression changes were statistically significant (P < 0.05). This demonstrates that TRIP4 binds to the hTERT promoter region from -322 to -902, proving that it is a transcription factor promoting hTERT transcription in cervical cancer.We discovered that knocking down TRIP4 was followed by a reduction in hTERT expression when TRIP4 siRNA was utilised to transfect HeLa and SiHa cell lines. TRIP4 overexpression, on the other hand, enhanced hTERT expression. The clone creation experiment was conducted after TRIP4 overexpression or hTERT gene silencing was performed on the HeLa cell line in subsequent studies. It was observed that the cells without overexpression and hTERT silencing showed radiosensitization and dose dependence. In order to determine whether TRIP4 regulates the expression of the hTERT protein, the next-step experiment was performed to stabilize the expression of TRIP4 in HeLa cells. We transfected it with a luciferase plasmid driven by the hTERT promoter and measured it after 48 hours of treatment with luciferase expression. It was observed that knockdown significantly inhibited the expression of luciferase driven by the hTERT promoter. Then, we used the DNA-protein assay (pulldown) and chromatin immunoprecipitation assay (CHIP) to further confirm the binding of TRIP4 to the hTERT promoter. We mix streptavidin-agarose beads, biotin-labeled hTERT promoter probe, and nucleoprotein from HeLa cells to stably express the control vector or TRIP4. As a result, it was found that the TRIP4 protein was detected by Western blotting in the pulldown complex. To validate the aforementioned findings, we created six luciferase reporter vectors with various lengths of hTERT promoters. In addition, we cotransfected HeLa cells with a plasmid expressing TRIP4 and a luciferase reporter gene controlled by the hTERT promoter or cotransfected cells with LacZ and TRIP4-luciferase plasmids. The expression of luciferase in cells cotransfected with TRIP4 and hTERT  luciferase plasmids was higher than that of hTERT promoter regions of other lengths (We investigated the P13K/AKT and MAPK/ERK pathways in cervical cancer HeLa cell lines and SiHa cell lines in order to identify the particular mechanism of TRIP4 on cervical cancer, and we predicted that decreasing the production of the TRIP4 protein might block this route. The findings of the experiment show that the hypothesis is valid. TRIP4 may activate the P13K/AKT and MAPK/ERK pathways and control the growth of cervical cancer via this route, according to knockdown and overexpression studies. Furthermore, we discovered that blocking this pathway may have an antitumor impact by reducing TRIP4 protein production and its underlying mechanism. Moreover, investigations have shown that when TRIP4 is knocked down, the expression of the hTERT protein decreases, and when TRIP4 is overexpressed, the expression of the hTERT protein rises again. Furthermore, when TRIP4 is knocked down or overexpressed, the hTERT protein is knocked down at the same time, reducing the survival curve of cervical cancer cells and increasing radiosensitivity. The binding relationship between TRIP4 and hTERT proteins, as well as the extent of TRIP4's regulatory sites on the hTERT gene, was determined using a luciferase test. Furthermore, the findings of this study show for the first time that knocking down TRIP4 reduces cell viability by inhibiting the P13K/AKT and MAPK/ERK pathways, which corresponds to the first part of the experimental results of colony formation inhibition and increased apoptosis in HeLa and SiHa cells. Inhibition of TRIP4 and hTERT proteins may also enhance the radiosensitivity of cervical cancer cells, according to this study. These results suggest that inhibiting TRIP4 may be a new therapeutic approach for cervical cancer therapy that specifically targets the P13K/AKT and MAPK/ERK pathways as well as the hTERT pathways in cervical cancer cells.