A sharp, intense band at 1028 cm−1 was observed in the Raman spectra of the nanoparticles. The shift of this band in comparison to AB itself agrees well with the theoretical model. AB in the nanoparticles was identified by means of electrochemistry and NMR spectroscopy. The sizes of the Au crystallites measured by XRPD were about 9 and 17 nm for the nanoparticles obtained in pH 7.4 and 3.6, respectively. The size of the particles as measured by TEM was 24 and 30 nm for the nanoparticles obtained in pH 7.4 and pH 3.6, respectively. The DLS measurements revealed stable, negatively charged nanoparticles.The aim of our work was the synthesis and physicochemical characterization of a unique conjugate consisting of gold nanoparticles (AuNPs) and a pharmacologically active anticancer substance abiraterone (AB). The direct coupling of AB with gold constitutes an essential feature of the unique AuNPs–AB conjugate that creates a promising platform for applications in nanomedicine. In this work, we present a multidisciplinary, basic study of the obtained AuNPs–AB conjugate. Theoretical modeling based on the density functional theory (DFT) predicted that the Au Abiraterone (AB) is administered as an acetate ester prodrug which is rapidly converted in vivo to abiraterone . AbirateChallenges related to modern drug form technology consist, among other things, in using techniques and technologies that allow us to deliver the drug directly to the drug target, extend the time of the API’s  activity in the drug target, and influence the API’s distribution. In the light of the above, nanotechnology gains primary importance ,3,4,5. I8, Au20 with alanine, tryptophan), (1+) conjugate the geometry where the NH terminal is located nearby the Au5 cluster is energetically more favourable than the geometry with the OH terminal in front of Au5. However, the interaction energy shows an opposite trend, i.e., the binding energy of Au5 in the [Au5–(NH)abiraterone)](1+) conjugate is weaker than in the [Au5–(OH)abiraterone](1+) conjugate.The interaction energies, also called the binding energies (BE), fall into an interval of about 20 ± 5 kcal/mol (84 ± 21 kJ/mol), which is close to the values obtained in the course of similar investigations of the Aukcal/mol , Au13–guA diffractogram of the lyophilized mixtures obtained in pH 3.6 and 7.4 is characterized by sharp peaks in the range of 5–35° and broad as well as sharp peaks in the range of 35–85° . The ideUpper window: diffractograms of the mixtures, Au and NaCl peaks are indicated. Below: a simulated diffractogram of abiraterone form I. Insert: a magnification of the low intensity peaks range .Diffractograms of two AuNPs–AB conjugates obtained in pH 3.6 and pH 7.4 are compared in 13–(N)AB and Au13–(OH)AB model nanoconjugates in the range of 1800–1000 cm−1. One can see that substantial differences are visible in the ranges of 1660–1580 cm−1 and 1100–1000 cm−1.−1 comes from the stretching vibration of the C=C (B ring) bond. In the spectra of the AB molecule and the Au13–(OH)AB model nanoconjugate, the bands at 1667, 1640, and 1612 cm−1 originate mainly from the C=C (D ring) bond stretching vibrations and the pyridine ring stretching vibrations. However, in each band the participation of individual vibrations of the C=C (D ring) bond and the pyridine ring is different, which has been summarized in 13–(N)AB model nanoconjugate, three characteristic bands are observed in the range 1660–1580 cm−1: 1664, 1641, and 1617 cm−1. The first two bands originate mainly from the C=C (D ring) bond vibrations and the pyridine ring stretching vibrations. These bands are much more intense than their counterparts from the AB and Au13–(OH)AB spectra. Just as in the AB and Au13–(OH)AB spectra, the third band at 1617 cm−1 has small cm−1 intensity and originates from the vibration of the entire AB molecule.In the theoretical spectra of the AB molecule and both model nanoparticles, the band at 1740 cm−1, two isolated bands at about 1080 and 1040 cm−1 are observed in the spectra of AB and the Au13–(N)AB model nanoconjugate. The first one comes from the whole AB molecule vibrations in both spectra. The second band comes mainly from the pyridine ring breathing vibrations and the steroid moiety vibrations in the spectrum of the Au13–(N)AB model nanoconjugate. This band is much more intense than its counterpart from the AB and Au13–(OH)AB spectra. In the spectrum of the Au13–(OH)AB model nanoconjugate, three bands at 1039, 1052, and 1076 cm−1 are visible. These bands originate from the whole AB molecule vibrations (1076 cm−1), steroid moiety vibrations (1052 cm−1), and mainly the pyridine ring with the steroid moiety vibrations (1039 cm−1). The description of the spectra has been summarized in In the range of 1100–1000 cm13–(N)AB model nanoconjugate spectrum can indicate that the interactions between the Au13 cluster and the AB molecule occur via the N atom.A significant increase in band intensities in the AuAccording to our studies, abiraterone appears in three polymorphic forms: I, II, and III. In −1 and one characteristic intense narrow band at 1028 cm−1. The Raman spectrum of the nanoparticle obtained in pH 3.6 is characterized by broad bands at about 1658, 1590, 1565, 1534, 1509, 1463, 1415, 1355, 1295, 1260, 1242, 1223, 1186, 1160, 1096, and 1057 cm−1 and one characteristic intense narrow band at 1028 cm−1. This comparison shows that more broadened bands are present in the spectrum of the nanoparticle obtained in pH 7.4. This corresponds well to the smaller Au crystallites estimated by XRPD.13–(N)AB as well as from three abiraterone forms, in the ranges of 1660–1580 cm−1 and at 1100–1000 cm−1, have been collected. One can see that the bands in the nanoparticle spectra from the range of 1660–1580 cm−1 are shifted towards lower wavenumbers in comparison to the Raman spectra of forms II and III. However, the band at 1028 cm−1 visible in the nanoparticle spectra is shifted towards higher wavenumbers in comparison to its counterpart from the spectra of forms I, II, and III .In −1 and the presence of an intense band at 1028 cm−1 in both nanoparticle spectra can suggest that the interactions between the AB molecule and the AuNPs are via the N atom. These studies also prove that the pH changes influence the Au crystalline size in the nanoparticles and bands broadening in the AuNPs–AB spectra.The shift into lower wavenumbers in the range of 1660–1580 cmThe amount of AB on the AuNPs surface was measured using a thermogravimetric analysis . The TGA1H NMR spectrum of the abiraterone acetate nanoparticles was obtained. Signals appeared at approx. 4.43, 5.38, 6.10, 7.33, 7.78, 8.41, and 8.57 ppm. They fit very well with the signals recorded for abiraterone acetate: 4.40 (CH-3), 5.37 (CH-6), 6.10 (CH-16), 7.34 (CH-24), 7.75 (CH-25), 8.40 (CH-23), and 8.56 (CH-21) ppm. This proves that the particles of abiraterone acetate are present in the tested nanoparticle sample. The 1H NMR spectra of gold nanoparticles combined with abiraterone were recorded for various synthesis methods. The spectra were obtained with the signals, among others, at approx. 5.29, 6.10, 7.33, 7.75, 8.40, and 8.55 ppm. This matches very well the data obtained for abiraterone measured under similar conditions, where the following signals were obtained: 5.29 (CH-6), 6.10 (CH-16), 7.33 (CH-24), 7.75 (CH-25), 8.55 (CH-23), and 8.56 (CH-21) ppm. This clearly proves that abiraterone is present in the tested samples of the nanoparticles. It is worth noting that in the observed range of the chemical shifts, there is a difference in the chemical shift of the proton of the CH-6 group between abiraterone nanoparticles (5.29 ppm) and abiraterone acetate nanoparticles (5.38 ppm).Initially, the 1H NMR spectra of the nanoparticles  oxidation of cholesterol in the cyclohexanol structure, which was manifested by the presence of a peak at the potential of 1.9 V in the acetonitrile solution using a GCE electrode [Electrochemical experiments were carried out using differential pulse voltammetry (DPV) to confirm the formation of gold nanoparticles modified with abiraterone. Electrochemical tests were carried out in a DMSO solution containing 0.1 M TBAHFP . At the lectrode ,28. ThatWe have reported a novel method for the successful conjugation of abiraterone and gold nanoparticles, creating a unique nanoconjugate with expected anticancer properties and biomedical applications.-synthesis optimization and purification of the AuNPs–AB conjugates that would afford desirable-sized AuNPs,-theoretical modeling of the interactions of the Au clusters with AB,-development of the analytical methods for the AB identification in the AuNPs–AB conjugates and the AB quantitation in the supernatants,-development of analytical methods to study the nanoparticle formation mechanism,-development of the quantitative methods to estimate the covering of the gold nanoparticles by the AB substance,-development of an analytical methodology for the physicochemical characterization of the obtained nanoparticle at pH 3.6 and pH 7.4.The study comprised as follows:n at the N-side. The binding energy is smaller, of about 10 kcal/mol (42 kJ/mol), when abiraterone or abiraterone acetate faces Aun at the OH/C=O side.Roller-like abiraterone or abiraterone acetate molecules were predicted to form conjugates with small Au clusters at the N-side that would be more stable than those formed at the OH/C=O-sides by about 10 kcal/mol (42 kJ/mol). The binding energy in the conjugates of small Au clusters with abiraterone is predicted to be about 20 kcal/mol (84 kJ/mol) when abiraterone faces AuAB in the gold nanoparticle was identified by electrochemistry and NMR spectroscopy.The obtained nanoparticles were characterized by the negative zeta potential from −11 to −13 mV, which can suggest their high stability in water and likely low toxicity for normal cells.The XRPD technique proved the presence of the AuNPs in the lyophilized mixtures as well as in the nanoparticles obtained in pH 3.6 and 7.4. The average size of the Au crystallites estimated from the Scherrer formula was about 9 and 17 nm for the nanoparticles obtained in pH 7.4 and 3.6, respectively. A comparison of the AuNPs–AB precipitates obtained for two different pH values suggests a higher stability of the pH 7.4 conjugate as well as a smaller nanoparticle size as measured by TEM, but the measurements performed by the DLS technique proved that the diameter of the nanoparticle obtained in pH 7.4 is higher than that of the nanoparticle obtained in pH 3.6. In pH 7.4, the nitrogen atom participates in the hydrogen bonds and aggregation may take place: but this, however, requires further research.The AuNPs–AB conjugates are considered to be candidates for further study and possible application in anticancer therapy.


Molecular surveillance of newly diagnosed HIV-infections is important for tracking trends in circulating HIV-variants, including those with transmitted drug resistances (TDR) to sustain ART efficacy.Dried serum spots (DSS) are received together with the statutory notification of a new diagnosis. 'Recent infections' (<155 days) classified by a 'recent infection test algorithm'  are genotyped in HIV-protease (PR), reverse transcriptase (RT) and integrase (INT) to determine the HIV-1 subtype, to calculate prevalence and trends of TDR, to predict baseline susceptibility and to identify potential transmission clusters for resistant variants.ptrends < 0.01). The subtype A increment is mainly due to transmissions within men who have sex with men (MSM) while subtype C transmissions are associated with heterosexuals and people who inject drugs. The prevalence of TDR was stable at 11.0%  over the study period. Resistances to nucleotide RT inhibitors (NRTI) and PR inhibitors (PI) were 4.5% and 3.2%, respectively, without identifiable trends. In contrast, resistances to non-NRTIs  doubled between 2014 and 2016 from 3.2% to 6.4% (ptrend = 0.02) mainly due to the K103N mutation  predominantly detected in recently infected German MSM not linked to transmission clusters. Transmitted INSTI mutations were present in only one case (T66I) and resistance to dolutegravir was not identified at all. Reduced susceptibility to recommended first-line therapies was low with 1.0% for PIs, 1.3% for NRTIs and 0.7% for INSTIs, but high for the NNRTIs efavirence (4.9%) and rilpivirine (6.0%) due to the K103N mutation and the polymorphic mutation E138A. These trends in therapy-naïve individuals impact current first-line regimens and require awareness and vigilant surveillance.Between January 2013 and December 2016, 1,885 recent infections were analysed regarding the PR/RT genomic region, with 43.5% of these also being subjected to the analysis of INT. The proportion of HIV-1 non-B viruses  increased from 21.6% to 36.0%, particularly the subtypes A (5.0% to 8.3%) and C (3.2% to 7.7%; all  HIV infection is still a major public health concern in EU/EEA countries with approximately 30,000 new cases reported each year . PreventTo predict TDR in cART-naïve patients, the World Health Organization (WHO) defined relevant resistance mutations selected by protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs). These are summarized in the WHO surveillance drug resistance mutations (WHO SDRM) list from 2009 . Based oThe surveillance of transmitted resistances of integrase strand transfer inhibtors (INSTI) is of particular interest. Raltegravir was approved in Europe in 2007, followed by elvitegravir (2012) and dolutegravir (2014). The general tolerance and the low likelihood of resistance selection due to a high genetic barrier  led to tIn 2013 a molecular surveillance program was initiated in Germany that is based on the examination of viral sequences from recently infected among newly diagnosed HIV cases , 19. Thehttps://www.rki.de/DE/Content/InfAZ/H/HIVAIDS/Studien/InzSurv_HIV/beteiligte_Labore.html) was established that send along with the report form, residual serum from newly diagnosed HIV cases spotted onto a filter card (Whatman 903 filter paper) as dried serum spots (DSS). According to §13 of IfSG the RKI is authorized to receive blood residuals from diagnostics for surveillance purposes.According to the “Protection Against Infection Act”  of 2001, diagnostic laboratories in Germany are obligated to report newly diagnosed HIV infections anonymously to the German public health institute . For surveillance programs a network of approximately eighty diagnostic laboratories  including results from the BED IgG Capture EIA  and clinical data from the HIV-notification database  . Only repol-sequence. In cases where a subtype or circulating recombinant form (CRF) could not be assigned, a maximum-likelihood tree with bootstrap (IQ-TREE 1.5.5) was calculated using the HIV-1 subtype reference panel from the Los Alamos HIV sequence data base. Only subtype classifications based on bootstrap values of >70% in the tree topology were taken into account, otherwise they were classified as unique recombinant form (URF).HIV-1 genotypes from the protease (PR) and reverse transcriptase (RT) genomic region 2013–2016) and the integrase (INT) genomic region (2014–2016) were generated according to the previously published protocols 13–2016 a. Since 2https://hivdb.stanford.edu/pages/SDRM.worksheet.INI.html; updated in June 2016). Phenotypic resistance was predicted using the Stanford HIV Drug Resistance Database 8.4 algorithm (Stanford HIVbd) [The prevalence of TDR was calculated from the number of persons infected with viral variants carrying at least one mutation included in the WHO SDRM list . Transmid HIVbd) . Three ld HIVbd)  which inhttps://github.com/kavehyousef/code) [Drug resistance mutations present in a proportion of ≥ 0.5% in the dataset were defined to be `frequent mutations´ and were used for trend analysis. In addition, sequences carrying one of the `frequent mutations´ were applied to phylogenetic analysis to allow the spread of resistance mutations within transmission networks to be mapped. For this purpose sequences were aligned with 33 reference sequences from the Los Alamos database and trimmed to 1026 base pairs. To select the optimal tree model, Maximum Likelihood (ML) phylogenies were reconstructed using the Ultrafast Bootstrap approximation in IQ-TREE with 10,000 replicates with the integrated model selection algorithm . A groupef/code)  was usedef/code) , 30. The2 test was used for bivariate comparison and logistic regression to assess the odds ratios (OR) and 95% confidence intervals (CI). Changes in the prevalence over time were analyzed using the chi2 test for trend of odds.Statistical analyses were performed using STATA (version 14.2). Continuous variables were analyzed using median and interquartile range (IQR). The chiBetween 2013 and 2016, a total of 10,643 DSS of newly diagnosed HIV-cases were submitted to the RKI along with the anonymous report and 3,380 (31.8%) were classified as recent infections. From these we were able to obtain 1,885 (55.8%) HIV-1 genotypes of the PR and RT genomic regions. The median plasma viral load was 140,500 copies/ml , the median CD4 cell count was 454 cells/μl (IQR 304–612) and the median age of the newly diagnosed individuals was 36.3 years. Baseline patient characteristics are shown in HIV-1 genotypes of the INT genomic region were available for 820 of the 1,885 DSS (43.5%) from newly diagnosed cases in 2014 , 2015  and 2016 . The proportional distribution in subgroups was congruent between the total study population  and the subset (n = 820) .ptrend = 0.001). This trend is primarily based on the two above mentioned subgroups and can also be observed in non-Germans   and primarily affected MSM and persons of German origin , Table 2 > 0.05) .C = 40 and nAG = 48) largely originated from Sub-Saharan Africa (nC = 26 and nAG = 21) (ptrend = 0.008) and C (ptrend = 0.002)  the most prevalent were subtype A (8.2%), CRF02_AG (5.7%) and subtype C (5.0%) . Almost AG = 21) . The inc= 0.002) . While s= 0.002) .ptrend = 0.68) (ptrend = 0.47), while NNRTI resistance increased within the study period (ptrend = 0.06). This increase was significant between 2014 and 2016 (ptrend = 0.02). PI resistance did not show any tendency to increase or decrease between 2013 and 2016 (ptrend = 0.99)  . There w = 0.68) . Mono re = 0.68) . A trend = 0.99) .p = 0.03) and in Germans compared to non-Germans . However, the prevalence was also high in persons with American and African origin, but the total number of cases with this origin was low  (p < 0.01). However, the highest level of TDR (14.9%) was identified in subtype A (Significantly higher proportions of TDR were found in MSM compared to HET (ctively) . Moreoveubtype A .The thymidine analogue mutations (TAMs) M41L, K219Q and T215 revertants as well as the non-TAM M184V were among the most frequently transmitted NRTI-resistance mutations (≥ 0.5%) . These Tptrend = 0.07) with this increase being significant between 2014 and 2016 (ptrend = 0.03). The steep slope of the `total K103N´ between 2014 and 2016 was quite congruent in the subgroups of MSM, persons of German origin and subtype B infections , namely the major primary mutation T66I resulting in high level resistance to elvitegravir and low level resistance to raltegravir. According to predictions from the Stanford HIVdb, phenotypic INSTI resistance  was identified in 0.7% (6/820) of cases  due to tSequences that had one or more of the most frequent NRTI, NNRTI and PI resistance mutations or the polymorphic E138A (listed in p = 0.67). Among the `not cluster-linked´ sequences carrying the K103N mutation the diversity of HIV-1 subtypes  as well as the origin of infected persons  was much higher. The proportion of individuals not linked to K103N-clusters was generally higher in 2015/2016 than in 2013/2014 (p = 0.01), although the sampling was more dense in 2015/2016 . Especially the group of `male persons´ and here particularly those for which the transmission route was not reported was significantly higher in 2015/2016 (p = 0.008) (p > 0.05)  , Fig 5. = 0.008) . However > 0.05) .In this study we analysed 1,885 recent HIV-1 infections newly diagnosed between 2013 and 2016. The proportions of HIV-1 subtypes and primary resistances in the total population and within subgroups  were stratified according to the year of diagnosis to allow analysing changes over time to be.In Germany, subtype B continues to be the predominant subtype (68.9%), although the proportion of non-B subtypes significantly increased between 2013 and 2016, particularly for subtypes A and CRF02_AG which peaked in 2015 (11.7% and 7.1%) and subtype C which doubled from 3.6% in 2013/2014  , and the PI resistance mutations M46I and V82L. Nevertheless, the proportions of transmitted NRTI and PI resistance remained stable during the study period. NRTI resistance resulted mainly from the high prevalence of persisting TAMs. Many studies have shown by phylogenetic analysis that onward transmission among drug-naïve patients is the major reason for the maintenance of stable NRTI resistance levels –45. HoweTherefore, the predicted resistance to the currently recommended first-line regimens  consistiIn 2017, tenofovir-containing pre-exposure prophylaxis  was introduced in Germany. HIV-infection despite PrEP, due to subclinical drug levels or infection with resistant variants, might occur and fuel the emergence of TDR. So far, resistance mutations selected by tenofovir and emtricitabine  have been rare (below 1%). However, as MSM transmission networks have been identified to be a major source for the spread of HIV resistance by others  and by uFor the first time, we analysed, to which extent INSTI resistance was transmitted following the introduction of the first generation INSTI raltegravir in Germany in 2007. So far, the proportion of transmitted INSTI mutations is low with only one case identified. Phenotypic resistance to raltegravir and elvitegravir according to Stanford HIVdb is predicted to be 0.7% and resistance to the second generation INSTI dolutegravir was not detected at all. Primary drug resistance to INSTIs was also described to be rare in studies of European patients diagnosed in 2013  or 2015 One limitation of the present study is the relatively short study period. Changes over time should therefore be interpreted carefully. Furthermore, integrase genotyping only started in 2014, resulting in lower PCR success rates due to RNA degradation on DSS. A combined prediction including all drug classes was only possible for 43.0% of cases therefore, resistance to integrase was analysed separately in this dataset. Another limitation is that the SDRM list has not been updated since 2009 and resistance mutations to the newest drugs or drug classes (e.g. integrase inhibitors) had to be analysed with different mutation lists. Some of the SDRMs are only relevant to older drugs rarely used in today’s first-line regimens in EU/EEA countries . If the Despite effective cART, TDR is present at an overall stable proportion in Germany (11%). In particular, viruses carrying resistance mutations with low fitness cost are spread continuously by onwards transmission, and German MSM are generally driving the spread in Germany, both within transmission networks and outside of networks as shown for the K103N mutation. Intensified HIV-screening in these groups followed by early treatment with cART including the pre-treatment resistance testing as recommended in the current guidelines should help reduce the spread of resistant viruses in the ART naïve population. Due to the increasing use of INSTIs in first-line regimens and tenofovir/emtricitabin for PrEP, it is important to monitor TDR for public health efforts and in order to maintain the effectiveness of cART.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


To evaluate the effect of intramedullary nail and locking plate in the treatment of proximal humerus fracture (PHF).China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), Wan-fang database, Chinese Biomedicine Database (CBM), PubMed, EMBASE, Web of Science, and Cochrane Library were searched until July 2018. The eligible references all show that the control group uses locking plates to treat PHF, while the experimental group uses intramedullary nails to do that. Two reviewers independently retrieved and extracted the data. Reviewer Manager 5.3 was used for statistical analysis.Thirty-eight retrospective studies were referred in this study which involves 2699 patients. Meta-analysis results show that the intramedullary nails in the treatment of proximal humeral fractures are superior to locking plates in terms of intraoperative blood loss, operative time, fracture healing time, postoperative complications, and postoperative infection. But there is no significance in constant, neck angle, VAS, external rotation, antexion, intorsion pronation, abduction, NEER, osteonecrosis, additional surgery, impingement syndrome, delayed union, screw penetration, and screw back-out.The intramedullary nail is superior to locking plate in reducing the total complication, intraoperative blood loss, operative time, postoperative fracture healing time and postoperative humeral head necrosis rate of PHF. Due to the limitations in this meta-analysis, more large-scale, multicenter, and rigorous designed RCTs should be conducted to confirm our findings.PROSPERO CRD42019120508 PHF is the third common limb fracture, accounting for 4 to 5% of total body fractures . The incDue to the good biomechanical properties of the locking plate and the intramedullary nail, its exact clinical efficacy has become the main treatment –19. HoweA literature retrieve was carried out in eight databases from their inception to July 2018, like CNKI, VIP, Wan-fang database, CBM, PubMed, EMBASE, Web of Science, and Cochrane Library. Search terms including “Proximal humerus fracture,” “Intramedullary nail,” “Locking plate,” and “Internal fixation” were used individually or in combination. The publishing language was restricted to Chinese and English.The inclusion criteria are as follows: (i) internal fixation of displaced proximal humeral fractures; (ii) included both locking plates and intramedullary nails; (iii) greater than a minimum of 6 months of follow-up; (iv) a minimum of 21 patients for a given study; and (v) clinical outcomes during follow-ups included at least one of the following: intraoperative blood loss, operative time, fracture healing time, postoperative complications and postoperative infection, constant, neck angle, VAS, external rotation, antexion, intorsion pronation, abduction, NEER, osteonecrosis, additional surgery, impingement syndrome, delayed union, screw penetration, and screw back-out.The exclusion criteria are as follows: (i) non-humeral proximal fracture; (ii) treatment mode non-locking plate or intramedullary nail treatment; (iii) non-clinical researches, basic researches, and review articles were excluded, as case reports and theoretical discussions; (iv) improper statistical methods, data defect literature; (v) genetic research; (vi) grey literature; and (vii) letters to editor.Two investigators (Xiaoqing Shi and Hao Liu) independently extracted and screened the data according to the inclusion criteria. We extracted the general details, such as patients’ characteristics, interventions, and outcomes, and a cross-check was done. Any disagreements were resolved through discussion or verification by a third investigator (Runlin Xing).The quality of the non-randomized controlled trials was assessed by the MINORS entry, and trials with MINORS scores > 12 were included in the study . The metχ2 test and inconsistency index statistic (I2). If substantial heterogeneity existed (I2 > 50% or P < 0.05), a random effect model was applied; otherwise, we adopted a fixed effect model [Revman 5.3 software was employed to pool the effect size. Mean difference (MD) or standardized mean difference (SMD) and 95% confidence intervals (CIs) were used for continuous variables. For the two-category data, we used OR (odds ratio)/RR (risk ratio) and 95% CIs as the efficacy analysis statistic. Heterogeneity was evaluated statistically using the ct model . SensitiA total of 506 articles were initially obtained through the search strategy. After excluding 298 duplications, the remaining articles were screened based on their titles and abstracts, and 148 records were removed. By reading the full text, 14 literatures that did not meet the inclusion criteria were excluded. Finally, 38 trials –59 were There were a total of 2699 patients (1238 in the locking plate group and 1461 in the intramedullary nail group) enrolled in our studies. More details of the included studies were presented in Table I2 = 96%, P < 0.00001, and the heterogeneity was high. Therefore, the random effect model was used to calculate the combined effect. The results showed that intramedullary nail in the treatment of PHF is statistically significant, as its intraoperative blood loss is less than the locking plate [SMD = − 2.67, 95% CI , Fig. Twenty-two studies , 53–57 rI2 = 92%, P < 0.00001, and the heterogeneity was higher. Therefore, the random effect model was used to calculate the combined effect. The results showed that intramedullary nailing for the treatment of PHF was statistically significant in reducing surgical time compared with locking plates [SMD = − 1.59, 95% CI , Fig. A total of 878 cases in the experimental group and 1055 cases in the control group, in 26 studies –55, 57, I2 = 92%, P < 0.00001, and the heterogeneity was high. Therefore, the random effect model was used to calculate the combined effect. The results showed that intramedullary nailing for the treatment of PHF was statistically significant in reducing surgical time compared with locking plates [SMD = − 0.68, 95% CI , Fig. Twenty studies , 52–56 rI2 = 0%, P = 0.52, and there was no heterogeneity. Thus, the combined effect model was used to calculate the combined effect. The results showed that intramedullary nailing for the treatment of PHF was better than the locking plate in the incidence of complications [OR = 0.75, 95% CI , Fig. Complications were reported in 29 studies , 53–59, We also analyzed other outcome indicators. Detailed information was shown in Table For the methodological quality and risk of bias of RCTs, we used the Cochrane Handbook for Systematic Reviews of Interventions 5.2.0 for evaluation. The results showed that no studies used double blindness. On the other hand, for non-RCTs studies, we used MINORS to assess the methodological quality of the included studies. The results showed that the score interval was 13–18 points. Specifically, 13 points for five studies, 14 points for nine studies, 15 points for ten studies, 16 points for three studies, 17 points for seven studies, and 18 points for four studies. In general, this meta-analysis has qualitative limitations and most of the included studies had high risk of bias and low methodological quality.To further confirm the stability of the above outcomes, we replaced the fixed effect model with random effect model and excluded the most and least weighted trials. Comparing with previous results, the outcome exhibited no obvious difference which revealed that our study was robust and reliable. We mainly assessed the publication bias of overall complications Fig. . The resIn recent years, intramedullary nail and locking plate have been the main choices of internal fixation for PHF. From the biomechanics analysis, Edwards et al.  establisThe results of this meta-analysis show that (1) intramedullary nail in the treatment of PHF, intraoperative blood loss, operation time, fracture healing time, postoperative complications, and postoperative infection is better than locking plate treatment; (2) there were no significant differences in constant, neck angle, VAS, external rotation, antexion, intorsion pronation, abduction, NEER, osteonecrosis, additional surgery, impingement syndrome, delayed union, screw penetration, screw back-out between intramedullary nail, and locking plate in the treatment of proximal humeral fractures; (3) the screw back-out rate of the two-part fracture is better than the intramedullary nail in the locking plate; the shoulder anterior flexion angle intramedullary nail of the four-part fracture is better than the locking plate.In terms of follow-up constant score, the intramedullary nail was not superior to the locking plate, and the results were not statistically different. Some studies concluded that may be related to surgical techniques –71. In tPrevious studies have suggested that there is no difference in the time of fracture healing between the two internal fixations. Jiang pointed out that this bias may be related to that the research is not enough in this area . In our In terms of the incidence of screw penetration, Konrad et al.  found thThere was a statistically significant difference in the overall risk of postoperative complications between the two groups, which is different from previous evidence-based studies –17. In oThe limitations of this study are as follows: (1) this study cannot examine the use of surgical instruments by various subjects and evaluate the skill level and proficiency of the surgeon, which may cause clinical heterogeneity and affect the reliability of the meta-analytical strength and conclusion; (2) the type of study is retrospective analysis, and there is risk of selective bias, which may affect the authenticity and reliability of the research results; (3) the lack of clinical randomized controlled study, and the level of evidence is not high; (4) the doctor’s procedure is not completely unified, bringing a part of clinical heterogeneity; and (5) in all trials, the manufacturers of intramedullary nails and locking plates are different, and their quality is not the same.Clinical outcomes have the potential to improve with time because the rate of the postoperative index can change with time. Despite the shortcomings in this study, we still try to avoid the risk of bias during the analysis and try subgroup analysis. Sensitivity analysis showed that the study has good stability and clinical reference value.Additionally, there are fewer reliable randomized controlled trials included in this article. The level of evidence was reduced. It is difficult to control bias or confounding factors effectively. The evaluation efficiency may be reduced, and there may be publication bias, selection bias, implementation bias, and measurement bias. The inverted funnel plot shows that the included literature is basically within 95% CI. The article has certain reference value, but its results and applications should be treated with cautious attitude. If there are more clinical randomized controlled trials in this area, then a reliable conclusion will be drawn.Intramedullary nailing for the treatment of proximal humeral fractures, intraoperative blood loss, operation time, fracture healing time, overall complication, and postoperative infection is better than locking plate treatment. In the treatment of proximal humeral fractures, the intramedullary nail and the locking plate are both mature. Before the proficiency of the technique, considering the treatment of proximal humeral fracture with intramedullary nail can bring effective results, such as reducing the surgical trauma, protecting the blood supply of the fracture end, promoting fracture healing, and reducing the occurrence of postoperative complications, especially the occurrence of postoperative infection. The author believes that intramedullary nail treatment is a better choice in the strict control of surgical indications. However, because the quality of the literature included in this study is various, there is a risk of bias. This conclusion needs to be demonstrated by more well-designed, high-quality, large-sample, multi-center, randomized, double-blind controlled clinical trials. In addition, the study and discussion of increasing related complications are conducive to obtaining more rigorous and objective clinical evidence.


On January 26, 2021, this report was posted online as an MMWR Early Release.On December 7, 2020, local public health officials in Florida county A were notified of a person with an antigen-positive SARS-CoV-2 testAn estimated 1,700 in-person school days were lost as a consequence of isolation and quarantine of patients and contacts during this COVID-19 outbreak.The American Academy of Pediatrics interim guidance for return to sports specifically recommends against mask wearing during wrestling because of the choking hazard that face coverings could pose (At the time of the tournament, the 14-day cumulative COVID-19 incidence in county A, home to seven of the 10 participating high school teams, was 363 per 100,000 persons; 7.7% of tests for SARS-CoV-2 had positive results (


Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV. Adenovirus (AdV) was first described in 1953 by Rowe and coworkers as a cytopathological agent isolated from human adenoids , 3. HAdV4–7–10–16–24–in vitro  is an effective inhibitor of HAdV lytic egress. The procedure leading to the identification of nelfinavir is described in another study using an imaging-based, high-content screen of the Prestwick Chemical Library (PCL) comprising 1,280 mostly clinical or preclinical compounds , 101. Ne50]) values of 25.7 μM, as determined by cell impedance measurements using xCELLigence (50) of nelfinavir was 27.1 (50 = 10.01 μM) and the effective concentration yielding 50% inhibition (EC50) of fluorescent-plaque formation (EC50 = 0.37 μM). The data indicate that nelfinavir is an effective, nontoxic inhibitor of HAdV-C2 multicycle infection.A recent paper describes a full-cycle, image-based screen of 1,278 out of 1,280 PCL compounds against HAdV-C2-dE3B-GFP, where clopamide and amphotericin B were excluded due to precipitation during acoustic dispension into the screening plates . The scrLLigence . xCELLigLLigence , 104. Thwas 27.1 , as deteWe first tested if nelfinavir affected viral protein production. HAdV-C2-dE3B-GFP-infected A549 cells were analyzed for green fluorescent protein (GFP) under the control of the immediate early cytomegalovirus (CMV) promoter and the late protein hexon expressed after viral DNA replication at 46 h postinfection (hpi). The results indicate that nelfinavir had no effect on GFP or hexon expression at the tested concentrations, while the formation of fluorescent plaques was completely inhibited . This rets1) particles, which lack the L3/p23 protease due to the point mutation P137L in p23 /VI and pVII/VII proteins using previously characterized antibodies. There was no evidence for an increase of pVI or pVII in HAdV-C5 from nelfinavir-treated cells, in contrast to temperature-sensitive 1  robustly reduced the number of dead cells and strongly reduced the number of infected cells at up to 100 PFU/well . Remarka50 values compared to the parental virus, for example, 2.1 versus 66.8 with A549 cells, 8.9 versus 61.0 with HeLa cells, and 4.6 versus 55.2 with human bronchial epithelial cells (HBECs)  . These e (HBECs) . The datFinally, we performed immunofluorescence experiments with HAdV-C2-dE3B-GFP-infected A549 cells at 44 hpi . Under nViruses are transmitted between cells by three major mechanisms, cell free through the extracellular medium, directly from cell to cell, or in an organism by means of infected motile cells or fluid flow in blood or lymphoid vessels. This can result in far-reaching or mostly local virus dissemination  occurred in regular HAdV-C2-dE3B-GFP infections, we analyzed A549 cells infected with <1 PFU per well in 160 wells up to 8 dpi. Thirty-three wells developed a single plaque. Twenty-four of them were fast-emerging comet-shaped plaques, of which the donor cell (indicated by the pink arrows) disappeared at between 2 and 3 dpi , top. In50 values of nelfinavir were heterogeneous for different HAdV types, as determined in A549 cells (50s (>10) ranging from 12.22 (HAdV-C1) to 71.09 (HAdV-C2). Members of HAdV species A and D and most of the HAdV-B types showed intermediate (2 to 10) to low (<2) nelfinavir susceptibility, notably HAdV-B7 and -B11 with TI50s of <1. MAdV-1 and -3 also showed low susceptibility. Noticeably, a high susceptibility of HAdV-C was consistently observed in human lung epithelial carcinoma (A549) cells, human epithelial cervix carcinoma (HeLa) cells, immortalized primary normal human corneal epithelial (HCE) cells, as well as normal HBECs. The corresponding TI50 values were in the same range as those for herpes simplex virus 1 (HSV-1), for which nelfinavir was reported to be an egress inhibitor  and MAdV-3 in mouse rectum carcinoma CMT93 cells. To balance statistical significance and automated plaque segmentation, we first determined the optimal amount of inoculum and duration of infection for each virus and cell line. The resulting TInhibitor , 112.50 values) formed exclusively comet-shaped plaques. Viruses with low TI50 values, such as A31, B11, or D37, had a high fraction of round plaques even when infected with >1 PFU/well. This demonstrates that the slowly growing round infection foci observed by fluorescence microscopy gave similarly shaped lesions due to cytotoxicity, akin to the lytic comet-shaped foci. We conclude that HAdV types employ lytic cell-free and nonlytic cell-to-cell transmission modes and give rise to different plaque phenotypes.We finally examined the plaque morphologies in nonperturbed infections by immunofluorescence staining of the late proteins VI and hexon as well as by microscopic analyses of crystal violet-stained dishes for classical plaques . Virusestrans-Golgi network (TGN) (116––A phenotypic screen of the PCL identified nelfinavir as a potent postexposure inhibitor of HAdV-C2-dE3B-GFP plaque formation in cell culture . Nelfinark (TGN) , 115. NeTGN) 116––128. It Here, we demonstrate that nelfinavir inhibits the egress of HAdV particles without perturbing other viral replication steps, including entry, assembly, and maturation. Morphometric analyses of fluorescent plaques indicated that HAdV-C propagates by two distinct mechanisms, lytic and nonlytic. Lytic transmission led to comet-shaped convection-driven plaques, whereas nonlytic transmission gave rise to symmetric round plaques. Nelfinavir specifically suppressed the lytic spread of HAdVs, most prominently the HAdV-C types and -B14, but not other HAdVs such as A31 or D37. Incidentally, HAdV-C and -B14 replicate to considerable levels in Syrian hamsters, whereas other HAdV types do not , 130. WeThe molecular mechanisms underlying cell lysis in AdV infection are not well understood, largely due to the lack of specific assays and inhibitors. Single-cell analyses combined with machine learning have started to identify specific features of lytic cells, such as increased intranuclear pressure compared to nonlytic cells . Lysis iP value of 4.1 to 4.68 . The virus was generated by the exchange of the viral E3b genome region with a reporter cassette harboring enhanced green fluorescent protein (GFP) under the control of a constitutively active cytomegalovirus (CMV) promoter. It was grown in A549 cells and purified by double-CsCl-gradient centrifugation   .HAdV types A31, B7, B11, B14a, B16, B34, C1, C6, D8, D30, and D37 were kindly provided by the late Thomas Adrian  and were verified by DNA restriction analysis , 164. HA166–l-glutamine , and 1% (vol/vol) penicillin-streptomycin  and subcultured following phosphate-buffered saline (PBS) washing and trypsinization  biweekly. HBECs were maintained in endothelial-basal medium  and passaged 1:1 weekly following PBS washing and trypsinization. Cell cultures were grown under standard conditions , and the passage number was limited to 20. The respective supplemented medium is referred to as supplemented medium.A549  cells, HeLa  cells, and HBECs  were obtained from the American Type Culture Collection (ATCC) . HCE  cells were obtained from Karl Matter . CMT93 (mouse rectum carcinoma) cells were obtained from Susan Compton, Yale School of Medicine. A549, HeLa, HCE, and CMT-93 cell cultures were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM)  containing 7.5% (vol/vol) fetal calf serum (FCS) , 1% (vol/vol) Nelfinavir mesylate (CAS number 159989-65-8) powder was obtained from MedChemExpress LLC  and Selleck Chemicals . The compound was dissolved in dimethyl sulfoxide (DMSO)  at 100 mM and kept at −80°C or −20°C for long-term or working storage, respectively.2, and 95% humidity) in duplicates. The 16-well E plates have a gold-plated sensor array embedded in their glass bottom by which the electrical impedance across each well bottom is measured. The impedance per well, termed the cell index (CI), is recorded as a dimensionless quantity. The background CI was assessed following the addition of 50 μl supplemented medium to each well and equilibration in the incubation environment. After 30 min of equilibration, 9,000 A549 cells in 50 μl supplemented medium were added per well, and measurement was started.Impedance-based assays were performed using the xCELLigence system (Roche Applied Science and ACEA Biosciences) as described previously , 153, ac50 indicates the concentration of nelfinavir that caused a 50% impedance reduction compared to the solvent-treated cells. The TC50 was calculated by nonlinear regression of the solvent-normalized CI over the concentration of nelfinavir.For the quantification of nelfinavir toxicity, 50 μl of the supernatant was removed 18 h later and replaced with 2-fold-concentrated nelfinavir or DMSO solvent as the control dilution in supplemented medium . The control was supplemented medium. Impedance was recorded every 15 min over 5 days. The TC6 viral particles [VP]/well of HAdV-C2-dE3B-GFP and 2.68 × 106 VP/well of HAdV-C2-dE3B-dADP, corresponding to ∼30 PFU/well). The delay of infection-induced cytotoxicity was calculated as the time point at which the CI of the infected cells had decreased by 50% relative to its maximum. Data analysis was performed using GraphPad , and curve fitting was performed using three-parameter inhibitor concentration-versus-response nonlinear regression.For the quantification of nelfinavir effects on the cytopathogenicity of HAdV-C2-dE3B-GFP compared to HAdV-C2-dE3B-GFP-dADP infection, 50 μl of the supernatant was removed 18 h later and replaced with nelfinavir- and virus-supplemented medium. Twenty-five microliters of 4-fold-concentrated nelfinavir  or the corresponding DMSO solvent control dilution  in supplemented medium or supplemented medium only was added to 50 μl of medium containing cells. Additionally, 25 μl of a 4-fold-concentrated virus stock dilution was added  paraformaldehyde (PFA) and 4 μg/ml Hoechst 33342  in PBS. Cells were washed three times with PBS and stored in PBS supplemented with 0.02% N3 for infections with viruses harboring a GFP transgene. For wild-type (wt) viruses, cells were quenched in PBS supplemented with 50 mM NH4Cl, permeabilized using 0.2% (vol/vol) Triton X-100 in PBS, and blocked with 0.5% (wt/vol) bovine serum albumin (BSA) in PBS. Cells were incubated with 381.7 ng/ml mouse anti-HAdV hexon protein antibody  and subsequently stained using 2 μg/ml goat anti-mouse-Alexa Fluor 594 . Plates were imaged on either an IXM-XL or an IXM-C automated high-throughput fluorescence microscope  using a 4× objective in wide-field mode. Hoechst staining was recorded in the 4′,6-diamidino-2-phenylindole (DAPI) channel, the fluorescein isothiocyanate (FITC)/GFP channel was acquired for viral GFP, and the tetramethyl rhodamine isothiocyanate (TRITC)/Texas Red channel was acquired for hexon immunofluorescence staining.Per 96-well plate, 15,000 A549 cells, 10,000 HeLa cells, 30,000 HBECs, 30,000 HCE cells, or 30,000 CMT-93 cells were seeded in 100 μl of the respective supplemented medium and allowed to settle for 1 h at room temperature (RT) prior to cell culture incubation at 37°C with 5% CO50 (infected and treated cells), the TC50 , as well as the corresponding standard errors (SE) were determined using curve fitting in GraphPad  using three-parameter inhibitor concentration-versus-response nonlinear regression. The mean TI50 was calculated as the EC50/TC50 ratio of the means. The TI50 SE was calculated by error propagation.The infection phenotype for each well was quantified using Plaque2.0 . The numInfection, HAdV hexon immunofluorescence staining, and imaging were performed in technical quadruplicates, as described above for the microscopic plaque assay. Single nuclei (Hoechst) were segmented using CellProfiler . Median 4 and 0.25 mg/ml ruthenium red for 1 h. Following washing with 0.1 M cacodylate buffer (pH 7.36) and H2O, the samples were incubated in 2% (vol/vol) uranyl acetate at 4°C overnight. The samples were dehydrated in acetone and embedded in Epon as described previously  and fixed at 4°C in 0.1 M ice-cold cacodylate buffer (pH 7.4) supplemented with 2.5% (vol/vol) glutaraldehyde and 0.5 mg/ml ruthenium red for 1 h. Cells were washed with 0.1 M cacodylate buffer (pH 7.4) and postfixed at RT in 0.05 M cacodylate buffer (pH 7.4) supplemented with 0.5% (vol/vol) OsOeviously . Slices HAdV-C5 was amplified in medium containing 0, 1.25, or 3 μM nelfinavir for 4 days. Cells were harvested and disrupted by three freeze-thaw cycles. The cell debris was removed by freon extraction, and mature full HAdV virions were purified by two rounds of CsCl gradient ultracentrifugation . The proDouble-CsCl-gradient-purified HAdV particles were adhered to collodion and 2% (vol/vol) amyl acetate film-covered grids . Viral particles were negatively stained with 2% (vol/vol) uranyl acetate and viewed on a transmission electron microscope  at 100 kV. Images were acquired using a charge-coupled-device (CCD) camera .±Nelfinavir stocks) and a size standard  were size separated on a 12% acrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane. HAdV proteins were detected using primary antibodies, 1:10,000 R72 rabbit antifiber -ester signal. Thirty z steps with a 0.5-μm step size were acquired for each channel, and maximal projections were calculated. Image analysis was performed using CellProfiler  using a 40× objective  in confocal mode (62-μm pinhole). The DAPI channel was acquired for nuclear Hoechst staining, the FITC/GFP channel was acquired for viral GFP, the TRITC/Texas Red channel was acquired for immunofluorescence ADP staining, and the Cy5 channel was acquired for the Profiler . NucleusProfiler . Statist8 VP/well of double-CsCl-purified HAdV-C5±Nelfinavir stocks in 100 μl ice-cold supplemented medium and kept on ice for 30 min. Following a 15-min entry phase under standard cell culture conditions, the cells were fixed, and the nuclei were stained for 1 h at RT by the addition of 33 μl 16% PFA and 4 μg/ml Hoechst 33342  in PBS. Following the above-described immunofluorescence staining procedure, the cell-bound HAdV virions were stained using 9C12 mouse antihexon    and goat±Nelfinavir stocks at 50 to 0.001 pg/well of a BCA-based viral protein and incubated under standard cell culture conditions. Cells were fixed at 52 hpi, stained for HAdV hexon expression, and imaged according to the procedure described above for the image-based plaque assay. Images were quantified using Plaque2.0  on an IXM-XL epifluorescence microscope . GFP-positive infected cells were classified based on the median nuclear GFP intensity using automated image analysis by CellProfiler .Four hundred eighty thousand A549 cells were seeded per 6-well dish, inoculated with 1,100 PFU HAdV-C2-dE3B-GFP/well for 1 h at 37°C, washed with PBS, and detached by trypsin digestion. Infected cells were centrifuged and resuspended in fresh medium to remove any unbound input virus. Cells were seeded at 180,000 cells/12-well plate in medium supplemented with 1.25, 3, or 10 μM nelfinavir or the respective DMSO solvent control. Viral progeny in the cell monolayer and supernatant was harvested at the indicated times postinfection by three freeze-thaw cycles. The lysates were cleared by centrifugation and stored at 4°C until titration on naive A549 cells. PFA-fixed, Hoechst-stained cells were imaged at 44 hpi using a 4× objective on an IXM-XL epifluorescence microscope . GFP-positive infected cells were classified based on the median nuclear GFP intensity using automated image analysis by CellProfiler . The yiel-glutamine , 7.5% fetal bovine serum (FBS) , 1% nonessential amino acids , 1% 100 mM sodium pyruvate , 0.25 ng/ml Hoechst 33342 , and 1 μg/ml propidium iodide (PI) . Plates were imaged at the indicated times postinfection on an IXM-C automated high-throughput fluorescence microscope  using a 40× objective  in confocal mode (62-μm pinhole). The DAPI channel was acquired for nuclear Hoechst staining, the FITC/GFP channel was acquired for viral GFP, and the Cy5 channel was acquired for the PI signal. Thirty z steps with a 0.5-μm step size were acquired for each channel, and maximal projections were calculated.Infection was performed as described above for the microscopic plaque assay. Cells were incubated with an inoculum ranging between 10 and 2,560 PFU/well HAdV-C2-dE3B-GFP for 1 h at 37°C. Cells were washed with PBS and 100 μl phenol-free DMEM  supplemented with 1% penicillin-streptomycin , 1% 2) with a centroid located 600 px from the well rim were considered to exclude spatial limitations. Plaque roundness was calculated as 1 − eccentricity, as follows: roundness = 1 − (4π × area)/(perimeter2).Plaques were segmented in Plaque2.0 , and plaStatistical analysis was performed in GraphPad  using the nonparametric Kolmogorov-Smirnov test.87–101 antibody  for 5 min on ice. The supernatant and washing PBS were collected, and cells were pelleted by centrifugation at 16,000 × g for 5 min at 4°C. Lysates were scraped off and used to resuspend the pelleted cells. Following another centrifugation step, the supernatant was collected and stored at −20°C. Samples of 15 μl of the lysate were supplemented with SDS-containing loading buffer . Samples were denatured at 95°C for 5 min, and proteins were separated on a denaturing 15% acrylamide gel. Proteins transferred to a PVDF membrane were detected with a 1:1,000 dilution of a rabbit anti-HAdV-C2-ADP78–93 antibody -supplemented DMEM containing a 1:12 dilution of HAdV-C2/5-neutralizing dog serum   suppleme2 atmosphere. At the indicated times postinfection, cells were fixed and stained for 60 min with a PBS solution containing 3 mg/ml crystal violet and 4% PFA added directly to the medium from a 16% stock solution. Plates were destained in H2O, dried, and imaged using a standard 20-megapixel phone camera under white-light illumination.Plaque shapes were also assessed by a conventional crystal violet-stained plaque assay performed on A549 cells in liquid-supplemented DMEM. All infections were performed at 37°C with 95% humidity and a 5% CO


Heart failure (HF) is the final stage of various cardiac diseases with poor prognosis. The integrated traditional Chinese medicine (TCM) and western medicine therapy has been considered as a prospective therapeutic strategy for chronic heart failure (CHF). There have been small clinical trials and experimental studies to demonstrate the efficacy of Shenfu Qiangxin Pills (SFQX) for treating CHF, however, there is still a lack of further high-quality trial. This paper describes the protocol for the clinical assessment of SFQX in CHF (heart-kidney Yang deficiency syndrome) patients.A randomized, double-blind, parallel-group, placebo-controlled, multi-center trial will assess the efficacy and safety of SFQX in the treatment of CHF. 352 patients with CHF (heart-kidney Yang deficiency syndrome) from 22 hospitals in China will be enrolled. Besides their standardized western medicine, patients will be randomized to receive treatment of either SFQX or placebo for 12 weeks. The primary outcome is the plasma N-terminal pro-B-type natriuretic peptide levels, which will be measured uniformly by the central laboratory. The secondary outcomes include composite endpoint events , echocardiography indicators, grades of the New York Heart Association (NYHA) functional classification, the 6-minute walk test (6MWT) results, Minnesota Living With Heart Failure Questionnaire and TCM syndrome scores.The integrated TCM and western medicine therapy has developed into a treatment model in China. The rigorous design of the trial will assure an objective and scientific assessment of the efficacy and safety of SFQX in the treatment of CHF.Chinese Clinical Trial Registry: ChiCTR2000028777 . Over the last 3 decades, amelioration of treatments and implementation have improved survival and reduced the hospitalization rate in patients with heart failure with reduced ejection fraction (HFrEF). According to the guidelines for chronic heart failure (CHF) treatment, angiotensin receptor neprilysin inhibitor, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, beta-blockers, aldosterone receptor antagonists, diuretics, digitalis, and vasodilating agents are standard treatments for heart failure. However, the outcomes often remain unsatisfactory. Traditional Chinese medicine (TCM) has the characteristics of multi-target, multi-function and multi-pathway in the process of prevention and treatment of HF. The integrative treatment of western medicine and TCM for HF is associated with increased quality of life, low re-admission rate, and favorable prognosis.Heart failure (HF), as the final stage of various cardiac diseases, is an abnormality of cardiac structure or function.–8 Some small-scale clinical studies indicated that SFQX had the advantages of enhancing cardiac contractility and improving heart function.,10China guidelines for the diagnosis and management of heart failure published in 2014 and 2018 all cited evidence from clinical studies of TCM, so more attention has been paid to TCM and it has been widely used in clinical practice. Shenfu qiangxin pills , a patent drug made of Ginseng(Renshen), Radix aconiti carmichaeli (Fuzi), rhubarb (Dahuang), the root bark of white mulberry (Sangbaipi), Polyporus umbellata, Semen lepidii (Tinglizi), grifola (Zhuling), have the effects of nourishing Qi, warming Yang, strengthening heart, and promoting diuresis. The pills have been commonly used in TCM for the integrative treatment of patients with CHF who are also diagnosed with heart-kidney Yang deficiency syndrome characterized with palpitations, breathlessness, chest tightness, puffy face and swollen limbs. Experimental researches have shown in animal models that SFQX could reduce water-sodium retention, correct electrolyte disturbance, protect renal and cardiac function via attenuating autophagy and apoptosis. Hence, further rigorously designed randomized controlled trials are warranted to assess the effect of SFQX in patients with CHF so as to provide high-quality evidence for clinical practice.However, the explicit role of SFQX in preventing and treating cardiovascular disease remains unclear due to a lack of sound scientific evidence. Currently available randomized controlled trials on SFQX are flawed, with small sample sizes, making it difficult to draw definite conclusions on the actual benefits and harms of it.We would like to test the hypothesis that patients with CHF will benefit from SFQX and evaluate its safety through a high-quality clinical trial.22.1The main objective of this study is to evaluate the safety and efficacy of SFQX so as to provide high-quality research evidence for its clinical practice compared to placebo for treating CHF.2.2This study is designed as a randomized, double-blind, parallel-group, placebo-controlled, multi-center trial. There are 22 institution hospitals located in different areas of China in this trial. A total of 352 stable patients with CHF, who fulfill the inclusion and exclusion criteria, will be randomized into either an experimental or control group in the ratio of 1:1. The general design of this study is demonstrated in Figure 2.32.3.1Recruitment of the participants began in January 2020 and is expected to be finished in December 2021. Recruitment may be extended depending on registration completion. All participants will be given an informed consent form with a detailed explanation before enrollment, and notified that they may be withdrawn from the trial at any point without penalty. Participants can be recruited directly or openly during the clinical practice or through screening the database.2.3.2(1)Western medicine diagnostic criteria: refer to the 2018 guidelines for the diagnosis and treatment of heart failure in China.(2),12TCM syndrome diagnostic criteria : refer to the 2014 expert consensus on diagnosis and treatment of TCM in CHF and 2016 expert consensus on diagnosis and treatment of integrated traditional and western medicine in CHF.2.3.3(1)Participants who meet the diagnostic criteria of CHF, and have a history of CHF for more than 3 months or clinical symptoms of HF for more than 3 months.(2)Participants with heart-kidney Yang deficiency syndrome based on differentiation of syndromes and treatment.(3)Aged 18 to 80 years.(4)Left ventricular ejection fraction (LVEF) < 40%.(5)New York Heart Association (NYHA) class II to IV.(6)N-terminal pro-B-type natriuretic peptide (NT-proBNP) ≥450 pg/ml.(7)Participants who have received standardized western medicine treatment with the optimal therapeutic dose for at least 2 weeks.(8)Participants who have not used TCM for heart failure within 2 weeks before enrollment.(9)Submitted informed consent.2.3.3.1(1)Participants who have undergone coronary revascularization and cardiac resynchronization therapy within 12 weeks or cardiac resynchronization therapy planned within 12 weeks.(2)Participants with severe primary diseases such as liver and kidney hematopoiesis and so on; alanine transaminase, aspartate transaminase, alkaline phosphatase, and/or serum creatinine values that are 2 times higher than the normal upper limit; serum potassium content >5.5 mmol/L; patients with tumor, severe neuroendocrine diseases and mental illnesses, etc.(3)Participants with left ventricular outflow tract obstruction, acute myocarditis, hypertrophic cardiomyopathy, restricted cardiomyopathy, aortic aneurysm, arterial dissection, congenital heart disease, severe arrhythmia, and significant hemodynamic changes in unrepaired heart valve disease(4)Participants with uncontrolled hypertension, systolic blood pressure ≥180 mmHg and/or diastolic blood pressure ≥100 mmHg.(5)Participants with severe peripheral arterial disease, acute attack of chronic obstructive pulmonary disease, pulmonary vascular disease such as primary pulmonary hypertension or pulmonary hypertension due to autoimmune disease.(6)Participants who are pregnant or preparing for pregnancy and lactation.(7)Participants with an allergic constitution, known sensitivity to the study drugs or their ingredients.(8)Participants in other clinical trials within 1 months.2.4The participants who provide written informed consent and meet the inclusion and exclusion criteria will be selected at the screening visit, namely visit 1. Eligible participants will be informed via telephone call and will be asked to visit the trial center within 1 week (visit 1). At visit 1, the investigator will record demographic information, other medical history and medication, vital signs, physical examination and so on, and subjects will be randomized to the experimental or control group. The dosage used in this study is 5.4 g 2 bags) of SFQX or placebo 3 times daily for 12 weeks. Tests and assessments will be performed according to the following schedule: Visit 2 , TCM syndrome scores, composite endpoint events, complete blood count, routine urine test, liver function test, renal function test, serum electrolytes, 12-lead electrocardiogram); Visit 3 and Visit4 are the same as above. Detailed assessment schedules are outlined in Figure  bags of 2.5The shape and appearance of SFQX and placebo are the same: tan herbal pills. Each bag of pills is manufactured at a dosage of 2.7 g, with shelf-life of 18 months. The manufacturing company  follows the regulations for good clinical practice, and controls the quality of the products using their own standards and testing methods.The principle investigator will receive the products used for this trial from the company and supply them to the drug administrator. The drugs for each subject are in a box consisting of three packages (1 package for each visit period) which are labelled. A package, which includes 168 bags for 28 days plus 18 bags for spare will be prepared at 4-week intervals from visit 1 to 3. All products used in the trial will be recorded, including the amount of the medication, the date of the delivery, and the date of return.After randomization, the participants will begin taking 5.4 g (2 bags) of SFQX or placebo orally combined with standardized western medicine treatment 3 times daily after meals for 12 weeks.2.6The central random method is used in this trial where IWRS system is used for central randomization and the assignment scheme is hidden. A block dynamic randomization method is used for randomized allocation. Randomized code generation and drug blinding will be implemented independent of the data. The trial is double-blind where the placebo is the same as SFQX in shape, color, smell, properties, and so on. Both the participants and the investigators will be blinded until completion of the trial.2.7 We assume the proportion of patients demonstrating a decrease in NT-proBNP level by at least 30% in the treatment group would be 48%. Therefore, given a type I error rate of α = 0.05, a power of 80% (type II error rate of β = 0.20), the sample size of each group is 146 cases calculated by PASS11 software. Moreover, considering a dropout rate of approximately 20% for randomized patients, a total of 352 patients  is required to be randomized for the efficacy analysis.The sample size calculation is based on the proportion of patients with a decrease of NT-proBNP level by at least 30%. According to the reference, 47.95% of the patients in the qili qiangxin capsule group had reductions in NT-proBNP levels by at least 30%, compared with 31.98% of patients in the placebo group.2.8P < .05 is considered statistically significant. Statistics software SAS 9.4 will be used for the data analysis. Measurement data are analyzed by paired t test, variance analysis, rank sum test, and so on. Enumeration data are analyzed by chi-square test, Fisher exact test, etc. Ranked data are analyzed by Ridit or Cochran-Mantel-Haensel. The analysis data set will be selected from full analysis set, per-protocol set and safety analysis set. Considering the influence of baseline value, covariance analysis or logistic regression analysis will be used to deduct the baseline factor.The statistical significance of this study is conducted by 2-sided test and 2.92.9.1The primary outcome is NT-proBNP, Efficacy assessment of primary outcome is based on either the change in NT-proBNP level or the proportion of the patients with NT-proBNP level decreased by at least 30% from baseline to 12 weeks.2.9.2The secondary outcomes will include composite endpoint events (CCEs), echocardiography indicators , grades of NYHA functional classification, 6MWT results, MLHFQ, TCM syndrome scores. Efficacy assessment of secondary outcomes is based on the change in echocardiography indicators, grades of NYHA functional classification, 6MWT results, MLHFQ, TCM syndrome scores from baseline to 12 weeks and the proportion of patients with composite endpoint events at the end of the trial.2.10The safety assessment is based on spontaneous reports of adverse events, vital signs, laboratory tests. Vital signs include temperature, blood pressure and heart rate and breathing. Laboratory tests include complete blood count , routine urine test , liver function test , renal function test , serum electrolytes  and 12-lead electrocardiogram.Adverse events will be recorded at any time during the trial. If any serious adverse event occurs during the trial, the investigator shall immediately take appropriate treatment for the subject, and report to the medical ethics committee of the primary research institution and the sponsor.2.11Data collection and management consists of 2 parts: one is Case Record Form (CRF) and another is ResMan public platform in Electronic Data Capture (EDC). Medical information obtained from this trial will be recorded in each patient's CRF and remain confidential which will be checked with the original medical records of the subjects. The researcher's EDC user name and password are specially assigned to enter the data. With the data of each center institution completed and all questions resolved, the project manager and the main researcher need to review the contents of each case again. The data administrator makes a database lock list and saves data management related documents as required. All research data will be kept for 5 years which include confirmation of all subjects, original written informed consent, CRFs, detailed records of drug distribution, etc.2.12A specialized monitor will be responsible for supervising the entire trial process, regularly review and verify whether the trial is conducted and documented on the basis of plan, standard work guidelines, and relevant regulations.2.13This trial protocol was approved by the Ethics Committee of Xiyuan Hospital of China Academy of Chinese Medical Sciences in 12 December 2019 (2019XLA062-4). Any protocol deviations will be approved by the Ethics Committee of Xiyuan Hospital of China Academy of Chinese Medical Sciences. The trial has been registered with the Chinese Clinical Trial Registry. All participants or authorized surrogates will be given a detailed explanation on this trial with the informed consent form and appropriate time to determine consent or assent.3HFrEF is characterized by defects in cardiac contraction. Although the conventional therapeutic approaches in HFrEF management have improved survival rates in patients, the prognosis still remains poor. The efficacy of integrative treatment for CHF with TCM and western medicine has been gradually accredited and the integrative treatment has been considered as an alternative therapeutic strategy for CHF with fewer side effects.–20 have indicated that western medicine combined with SFQX could further improve cardiac function, TCM syndrome, the quality of life and show a clinical curative effect, improve LVEF and narrow left ventricular end diastolic diameter. They may work by reducing plasma brain natriuretic peptide and atrial natriuretic peptide, anti-inflammatory, inhibiting oxidative stress, protecting vascular endothelial function, suppressing rein-angiotensin-aldosterone system system, reversing ventricular remodeling to inhibit myocardial fibrosis. However, there is still a demand on a high-quality and larger-scale clinical research to prove its efficacy and safety in the treatment of CHF.As a post-marketed medicine, the composition of SFQX is clear and its quality is assured. Some clinical reportsThe study design of this trial has 3 main points.1.To assess the results of the treatment effectively, a standardized outcome measure is required. We select NT-proBNP as a primary outcome. Plasma will be collected in various hospitals and transported to the central laboratory  by cold chain for NT-proBNP detection. Plasma NT-proBNP levels were measured by dedicated kit-based NT-proBNP assays (Roche Diagnostics).2.CHF patients with heart-kidney yang deficiency syndrome will be included on the basis of syndrome differentiation and treatment, which reflects both the advantages and characteristics of TCM and the efficacy evaluation of western medicine. Patients with syndrome characterized by palpitations, shortness of breath or wheezing, fear of cold could be diagnosed.3.The study protocol was designed with reference to international clinical trial principles. Three-level quality control measures were taken and data were monitored by EDC system.Quality control supervisors will ensure that investigators adhere to the study protocol in each center institution. We used the random method of dynamic randomization (multiple blinding) of sub-center group to compete for the group, so as to manage data and reduce deviation in the research. The rigorous design of the trial will ensure an objective and scientific assessment of the efficacy and safety of SFQX in the treatment of CHF.We also thank the following coordination hospitals and directors:(1)Xiyuan Hospital of China Academy of Chinese Medical Sciences, Xiaochang Ma(2)Fuwai Hospital, Chinese Academy of Medical Sciences, Jian Zhang/Mei Zhai(3)Xuanwu Hospital Capital Medical University, Qi Hua(4)First Teaching Hospital of Tianjin University of TCM, Jingyuan Mao(5)Second Affiliated Hospital of Tianjin University of TCM, Yingqiang Zhao(6)Shuguang Hospital Attached to Shanghai TCM University, Xiaolong Wang(7)The First Affiliated Hospital to Changchun University of Chinese Medicine, Yue Deng(8)Shengjing Hospital of China Medical University, Zhijun Sun(9)Xinjiang Uygur Autonomous Region Hospital of Traditional Chinese Medicine, Xiaofeng Wang(10)Tianjin Chest Hospital, Shutao Chen(11)Guangdong Hospital of TCM, Weihui Lv(12)Affiliated Hospital of Jiangxi University of TCM, Zhongyong Liu(13)Affiliated Zhongshan Hospital of Dalian University, Qin Yu(14)Shijiazhuang No.1 Hospital, Xitian Hu(15)Handan First Hospital, Xianzhong Wang(16)Wuxi Traditional Chinese Medicine Hospital, Shu Lu(17)Huangshi central hospital, Daoqun Jin(18)Luoyang Hospital of TCM, Yanling Sun(19)TCM-integrated Hospital of Southern Medical University, Yiye Zhao(20)Yuncheng Central Hospital, Xia Wang(21)Xingtai People's Hospital, Hebei Medical University Affiliated Hospital, Limei Yao(22)Taiyuan Iron and Steel (Group) Co., Ltd., General Hospital, Qing JiThe hospitals all above are listed irrespective of ranking.Conceptualization: Lijun Guo, Dawu Zhang, Xiaochang Ma.Supervision: Xiaochang Ma, Jian Zhang, Qi Hua, Keji ChenWriting – original draft: Lijun Guo, Hui Yuan.Writing – review & editing: Lijun Guo, Hui Yuan, Xiaochang Ma.


Cervical cancer (CC) is the third most common gynecological malignancy around the world. Cisplatin is an effective drug, but cisplatin resistance is a vital factor limiting the clinical usage of cisplatin. Enhancer of mRNA decapping protein 4 (EDC4) is a known regulator of mRNA decapping, which was related with genome stability and sensitivity of drugs. This research was to investigate the mechanism of EDC4 on cisplatin resistance in CC. Two human cervical cancer cell lines, HeLa and SiHa, were used to investigate the role of EDC4 on cisplatin resistance in vitro. The knockdown or overexpression of EDC4 or replication protein A (RPA) in HeLa or SiHa cells was performed by transfection. Cell viability was analyzed by MTT assay. The growth of cancer cells was evaluated by colony formation assay. DNA damage was measured by γH2AX (a sensitive DNA damage response marker) immunofluorescent staining. The binding of EDC4 and RPA was analyzed by immunoprecipitation.EDC4 knockdown in cervical cancer cells (HeLa and SiHa) enhanced cisplatin sensitivity and cisplatin induced cell growth inhibition and DNA damage. EDC4 overexpression reduced DNA damage caused by cisplatin and enhanced cell growth of cervical cancer cells. EDC4 could interact with RPA and promote RPA phosphorylation. RPA knockdown reversed the inhibitory effect of EDC4 on cisplatin-induced DNA damage.The present results indicated that EDC4 is responsible for the cisplatin resistance partly through interacting with RPA in cervical cancer by alleviating DNA damage. This study indicated that EDC4 or RPA may be novel targets to combat chemotherapy resistance in cervical cancer. Cervical cancer (CC) is the third most common gynecological malignancy around the world. About one-third of the new cases are found in China, and showing an increasing incidence rate of young patients and early stages every year –3. AlthoEnhancer of mRNA decapping protein 4 (EDC4) is a known regulator of mRNA decapping to function in the mRNA P-bodies within the cytoplasm. Hazir Rahman found that EDC4 as an interacting partner of mTORC1, which is a rapamycin sensitive complex involved in the process of energy synthesis, translation, transcription, and lipid biosynthesis . EDC4 alp < 0.01 vs. control) in HeLa and SiHa cells, and sequence 2 was more potent for EDC4 knockdown. EDC4 depletion decreased the IC50 of cisplatin of HeLa (from 9.728 μM to 5.226 μM) and SiHa cells (from 25.29 μM to 9.423 μM), which indicated that the cancer cells were more sensitive to drug in terms of cell survival  by short hairpin RNA (shRNA) of two independent shRNA sequences (shEDC4#1 and shEDC4#2). The knockdown efficacy was confirmed by RT-PCR and Western blot. As shown in Fig. p < 0.05 for HeLa and p < 0.01 for SiHa), and EDC4 knockdown exhibited more potent inhibitory effect to the colony formation compared with cisplatin alone (shNC+DDP) (p < 0.01 for HeLa and p < 0.05 for SiHa). Besides, the DNA damage was measured by γH2AX (a sensitive DNA damage response marker) immunofluorescent staining. The results of γH2AX staining showed that cisplatin induced the amount of γH2AX positive cells  of HeLa and SiHa, and EDC4 knockdown obviously increased the intensity of γH2Ax  . Colony formation assay . Cisplatin decreased the amount of the colony of cancer cells, but was reversed by EDC4 overexpression . Besides, EDC4 overexpression did not induce the DNA damage, as the amount of γH2AX positive cells had no obviously changes. Cisplatin induced the increasing of γH2AX positive cells , while EDC4 overexpression inhibited the effects of cisplatin   were established. The transfection efficacy was confirmed by RT-PCR and Western blot. As shown in Fig. 01) Fig. e. These As EDC4 was related with cisplatin induced DNA damage, we further study that whether it interacted with RPA, a protein binds with ssDNA and responsible for DNA metabolism. To achieve the goal, the physical EDC4-RPA interaction was confirmed by co-immunoprecipitation assay . In accordance with the above results, EDC4 overexpression induced more colony formation of HeLa cells compared with negative control , while RPA1 or 2 knockdown reversed the effects of EDC4 overexpression on colony formation   Fig. b. Beside01) Fig. c. These Cervical cancer (CC) is the third most common gynecological malignancy around the world. Cis-Dichlorodiamineplatinum  is a commonly used and one of the most effective drugs for treatment of advanced or recurrent CC . HoweverEnhancer of mRNA decapping protein 4 (EDC4) is a known regulator of mRNA decapping to function within the cytoplasm and related with drug resistance. For example, researchers found that EDC4 deficiency of HeLa cells leads to hypersensitivity to DNA interstrand crosslinking drugs and PARP inhibitors, which also indicated the effects of EDC4 on DNA damage repair in the nucleus and chemo-sensitivity . In thisThe mechanism of cisplatin resistance is complex. Previous reports have demonstrated that modulating cellular response to DNA replication stress is a key factor of cisplatin resistance in cancer treatment . ReplicaIn conclusion, the present results indicated that EDC4 is responsible for the cisplatin resistance partly through interacting with RPA in cervical cancer by alleviating DNA damage. This study indicated that EDC4 or RPA may be novel targets to combat chemotherapy resistance in cervical cancer.2).Two human cervical cancer cell lines, HeLa and SiHa, were purchased from American Type Culture Collection  and cultured in DMEM medium supplemented with 10% fetal bovine serum in a humidified incubator  was used to establish the EDC4 knockdown cells. Human cervical cancer cell lines (HeLa or SiHa) were seeded in 6-well plates and subjected to transfection with EDC4 shRNA, or control shRNA using Lipofectamine 2000 according to the manufactory’s suggestion. To evaluate the effect of EDC4 overexpression, cells were transfected with EDC4 overexpressed plasmid or the negative control (Vector)  using Lipofectamine 2000 following the manufacturer’s protocol. The sequences of sh-RNAs were as follows: Sh-EDC4 #1:GGTGATAGTACCTCAGCAAAC; Sh-EDC4 #2:GCCACCCATTAACCTGCAAGA. The RPA knockdown was performed by small interfering RNA (siRNA)  and transfected to cells by Lipofectamine 2000. Briefly, HeLa or SiHa cells were loaded in 6-well plate and cultured until 80% confluence. Then, the premixed lipofection and plasmids were added into the wells and incubated for 24 h. The sequences of siRNAs for RPA1 and RPA2 were referred from the previous report .The cell viability was analyzed by MTT assay. Briefly, cells (EDC4 knockdown) were treated with cisplatin for 48 h, and MTT  solution (5 mg/mL) was added and incubated for another 4 h. DMSO was used to dissolve the formazan crystals. The absorbance at 570 nm was measured with a microplate reader . The IC50 was defined as the cisplatin concentration required inhibiting cell proliferation by 50%, and calculated by SPSS.The transfected or cisplatin treated cells were added in plates. After the incubation of 14 days, cells were washed twice with pre-cold PBS and stained with crystal violet.. The colony formation efficiency was represented as the percentage of colonies in seeded cells number. Experiments were repeated independently in triplicate.The immunoprecipitation was performed using Commercial immunoprecipitation kit  according to the manufactory’s protocol. Briefly, cells of different groups were trypsinized and collected. Cells were lysed, homogenized in lysis buffer and centrifuged 12,000 rpm to collect supernatant as cell lysate. For Co-IP experiments, cell lysate was incubated with purified antibody against target protein and incubated overnight at 4 °C. Then, Protein G-conjugated beads or IgG-conjugated magnetic beads were added to lysates and incubated for 3 h. Beads were pulled down magnetically and washed with IP buffer and PBS. After elution from beads, the bounding protein was analyzed by Western Blot.Cells were seeded in coverslips, fixed with 4% paraformaldehyde and permeabilized with Triton-100 (1%). After blocked with 5% goat serum, cells were incubated with primary antibody  and then with secondary antibodies conjugated to Alexa Fluor 488. Finally, the nuclei are labeled with DAPI . Immunofluorescence images were captured using FV10-ASW viewer software .Cells were lysed in RIPA buffer containing a protease inhibitor cocktail and quantified by BCA method. Then the protein samples were added with loading buffer and heat denatured at 94 °C. The equal amount of proteins was separated using 10% SDS PAGE and transferred to PVDF membranes. Then the membranes were blocked with BSA, incubated with primary antibodies : Abcam, ab109394) overnight at 4 °C, and finally incubated with the appropriate HRP-conjugated secondary antibodies. The protein levels were detected with ECL chemiluminescent system and the densitometry of blot was analyzed by ImageJ software.Total RNA was extracted and purified from cells by RNeasy Plus Universal Mini Kit  and reverse transcribed to cDNA in a 20 μL volume. Quantitative real-time PCR analysis was performed using SYBR Green q-PCR kit . The level of the different mRNAs was normalized to GAPDH. The primer sequences refered to published paper .p value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS software . Data was expressed as means ± SD. Significance was analyzed using two-tailed Student’s t test.