It remains uncertain if prior use of oral anticoagulants (OACs) in COVID-19 outpatients with multimorbidity impacts prognosis, especially if cardiometabolic diseases are present. Clinical outcomes 30-days after COVID-19 diagnosis were compared between outpatients with cardiometabolic disease receiving vitamin K antagonist (VKA) or direct-acting OAC (DOAC) therapy at time of COVID-19 diagnosis.A study was conducted using TriNetX, a global federated health research network. Adult outpatients with cardiometabolic disease (i.e. diabetes mellitus and any disease of the circulatory system) treated with VKAs or DOACs at time of COVID-19 diagnosis between 20-Jan-2020 and 15-Feb-2021 were included. Propensity score matching (PSM) was used to balance cohorts receiving VKAs and DOACs. The primary outcomes were all-cause mortality, intensive care unit (ICU) admission/mechanical ventilation (MV) necessity, intracranial haemorrhage (ICH)/gastrointestinal bleeding, and the composite of any arterial or venous thrombotic event(s) at 30-days after COVID-19 diagnosis.2275 patients were included. After PSM, 1270 patients remained in the study . VKA-treated patients had similar risks and 30-day event-free survival than patients on DOACs regarding all-cause mortality, ICU admission/MV necessity, and ICH/gastrointestinal bleeding. The risk of any arterial or venous thrombotic event was 43% higher in the VKA cohort .In COVID-19 outpatients with cardiometabolic diseases, prior use of DOAC therapy compared to VKA therapy at the time of COVID-19 diagnosis demonstrated lower risk of arterial or venous thrombotic outcomes, without increasing the risk of bleeding.The online version contains supplementary material available at 10.1186/s12933-021-01368-6. Oral anticoagulants (OACs), including vitamin K antagonists (VKAs) and direct-acting OACs (DOACs), have been used for thromboprophylaxis in different clinical scenarios. In the pivotal clinical trials of stroke prevention in atrial fibrillation (AF), DOACs were non-inferior to warfarin for preventing stroke/systemic embolism (SE), with lower rates of intracranial haemorrhage (ICH) in comparison with warfarin –4. SimilCoronavirus Disease 2019 (COVID-19) has shown to trigger endothelial dysfunction, inflammatory and hypercoagulable states –18. The The aim of this study was to compare clinical outcomes 30-days after COVID-19 diagnosis between outpatients with cardiometabolic disease on chronic VKA or DOAC therapy at time of COVID-19 diagnosis, using a propensity score matching (PSM) approach.Data from TriNetX, a global federated health research network with real-time updates of anonymised electronic medical records (EMRs) mainly from the United States (US), were used. The network includes healthcare organisations , with accumulated data for more than 71 million patients. Approximately, 18 million adult patients had a visit in a TriNetX HCO during 2020.The inclusion criteria were ≥ 18 years and outpatient with COVID-19 and cardiometabolic disease recorded in EMRs between 20 January 2020 and 15 February 2021. COVID-19 was identified using criteria provided by TriNetX based on Centers for Disease Control and Prevention (CDC) coding guidelines . COVID-1All patients were then stratified by OAC prescription. The DOAC group included outpatients who received either dabigatran, apixaban, rivaroxaban or edoxaban for at least 1 year before COVID-19 diagnosis, whereas the VKA group included outpatients who received warfarin under the same conditions. Patients were excluded if they received concomitant anticoagulant therapy . Baseline demographics, comorbidities and medication use were also captured from the patient EMRs.The searches were run in TriNetX on 30 April 2021, which allowed for at least 30-days of follow-up for all participants from the time all conditions were fulfilled. When the searchers were run, there were 61 participating HCOs within the TriNetX research network.All patients were followed-up for up to 30-days after COVID-19 diagnosis. Primary outcomes included all-cause mortality, ICU admission/mechanical ventilation (MV) necessity, ICH/gastrointestinal bleeding, and the composite of any arterial or venous thrombotic event . The secondary outcomes were hospital admission, myocardial infarction, VTE, ischemic stroke/TIA/SE, and all bleeding. Further details about the ICD-10-CM codes used for the identification of every outcome are included in Additional file As a federated network, research studies using TriNetX do not require ethical approval. To comply with legal frameworks and ethical guidelines guarding against data re-identification, the identity of participating HCOs and their individual contribution to each dataset are not disclosed. The TriNetX platform only uses aggregated counts and statistical summaries of de-identified information. No protected health information or personal data are made available to the users of the platform.t tests. Categorical variables were expressed as absolute frequencies and percentages, and tested for differences with chi-squared test.Continuous variables were expressed as mean and standard deviation (SD), and tested for differences with independent-sample The TriNetX platform was used to run 1:1 PSM using logistic regression. The platform uses ‘greedy nearest-neighbour matching’ with a caliper of 0.1 pooled standard deviations and difference between propensity scores ≤ 0.1. We assessed covariate balance between groups using standardised mean differences (SMDs). Any baseline characteristic with a SMD between cohorts lower than 0.1 is considered well matched .Cox proportional Hazard Ratios (HRs) with 95% confidence intervals (CI) for 30-days outcomes were calculated following PSM. Kaplan–Meier survival curves were also produced with Log-Rank tests after PSM. No imputations were made for missing data. Two-sided p-values < 0.05 were accepted as statistically significant. Statistical analysis was performed using the TriNetX Analytics function in the online research platform.Overall, 2275 patients  with COVID-19 and cardiometabolic disease were included. Of these, 648  were on VKA therapy at the time of COVID-19 diagnosis, and 1627  were on DOACs.There were no differences between cohorts regarding the main reasons for OAC (i.e. AF or previous pulmonary embolism). Other VTEs were more common in patients taking VKA therapy. In addition, patients on VKA were in general more comorbid, as demonstrated by the higher prevalence of hypertension, heart failure, ischemic heart disease, hyperlipidaemia, overweight/obesity, diseases of the nervous and digestive systems, acute kidney failure/chronic kidney disease, and neoplasms Table . After PIn the initial populations, all event rates were numerically higher in the COVID-19 patients with cardiometabolic disease on prior VKA, which were significant for any arterial or venous thrombotic event , VTE , and ischemic stroke/TIA/SE . After PSM, the rate of any arterial or venous thrombotic event  and ischemic stroke/TIA/SE  remained higher in users of VKA compared to users of DOACs. Further details are shown in Table In terms of the primary outcomes after PSM, there were no significant differences in the risks of all-cause mortality, ICU admission/MV necessity, or ICH/gastrointestinal bleeding between patients on VKA or DOACs. Thus, the risk for all-cause mortality was similar comparing the VKA cohort and DOAC cohort . The risk of ICU admission/MV necessity in the VKA cohort was not significantly different compared to the DOAC cohort , and the risk of ICH/gastrointestinal bleeding was similar . Event-free survival for these three outcomes was not different between cohorts, as assessed by the Kaplan–Meier analyses   and the lower event-free survival (Log-Rank test p = 0.029) . Likewise, there were no significant differences in the risk of myocardial infarction between both groups , nor in the risk of VTE . The risk of all bleeding events was not significantly different in patients previously taking VKA    with COVID-19, AF and other cardiometabolic disease were included, of which 433  were on VKA therapy at the time of COVID-19 diagnosis, and 433  were on DOACs.Overall, the risk for the primary outcomes in this sensitivity analysis between patients on VKA or DOACs was similar compared to the main analysis. Thus, there were no significant differences in VKA or DOAC-treated patients regarding all-cause mortality , ICU admission/MV necessity , and ICH/ gastrointestinal bleeding . The risk of composite outcome of any arterial or venous thrombotic event was higher in the VKA cohort compared to the DOAC cohort , with a significantly lower event-free survival (Log-Rank test p = 0.033).Concerning the secondary outcomes, the risks of hospital admission , myocardial infarction , VTE , and all bleeding events , was also similar in patients previously taking VKA or DOACs. The risk of ischemic stroke/TIA/SE was increased in VKA users , and Kaplan–Meier analysis demonstrated a significantly lower event-free survival in the VKA-treated cohort (Log-Rank test p = 0.006).outpatients with cardiometabolic disease, patients taking VKA before COVID-19 diagnosis showed a 43% higher 30-day risk of any arterial/venous thrombotic event and ischemic stroke/TIA/SE, compared to patients taking DOAC, adjusting for comorbidities using PSM. These results were consistent also in COVID-19 patients with AF and other cardiometabolic disease.In this study including COVID-19 Previous studies have shown that cardiometabolic multimorbidity is common among patients with COVID-19, and it is associated with a higher risk of hospitalization and a worsened prognosis –32. In pIt is well established that COVID-19 increases the risk of arterial and venous thrombosis , 21, leaNevertheless, a common limitation of most studies  is the hospitalization setting. Such patients have already suffered deterioration of their baseline status, which has led to hospitalization and may be a manifestation of a more severe state of the SARS-CoV-2 infection, with an increased risk of thrombosis, mortality, ICU admission, and MV. In these more severe patients, several confounding factors may be acting when evaluating the potential role of previous OAC, and the differences between VKA and DOAC may be non-existent.outpatients with cardiometabolic diseases under anticoagulation therapy are a population with scarce data, and their management could be particularly complex. A recent study concluded that OAC therapy in high-risk AF patients was associated with a lower risk of receiving a positive COVID-19 test and severe COVID-19 outcomes [As a result, COVID-19 outcomes . In turnoutcomes . Althougoutcomes , 48.DOACs have recently demonstrated to be superior in comparison to VKAs among patients with different conditions. For example, DOACs was associated with lower long-term all-cause mortality than VKAs in AF patients who were successfully discharged after transcatheter aortic valve replacement . In addiNonetheless, the published data for DOACs in the field of COVID-19 is conflicting. In the context of hospitalization, many of the COVID-19 patients with prior DOAC therapy will be switched to heparins because they are expected to receive medications interacting with DOACs, or have coagulation system and homeostasis disorders , 53. ForHowever, there are some indications such as antiphospholipid syndrome or prosthetic heart valves for which DOACs are not approved. In such patients, a switch from VKA to DOACs is not possible, and therefore other interventions are required to achieve and maintain the highest quality of anticoagulation in order to reduce the risk of worse clinical outcomes when on VKAs. These include the identification and modification of causes of poor anticoagulation control with VKAs , routine assessment of adherence to treatment, patient-centred education/counselling and educational programs for healthcare professionals, the use of multidose drug dispensing systems, self-management of VKA therapy, specialized anticoagulation clinics to improve adherence and a more careful follow-up , 58. DueThere are some limitations that need to be acknowledged. First, the data were collected from the HCO EMRs and some health conditions may be underreported. Recording of ICD codes in EMR may vary by factors including age, comorbidities, severity of illness, length of in-hospital stay, and in-hospital mortality. Further residual confounding may include lifestyle factors such as alcohol consumption and physical activity, which were not available.Propensity scores are a method used to balance covariates, but in observational studies propensity scores are estimated and therefore there is no certainty that the propensity score was 100% accurate. We also could not determine if there was any impact of attending different HCOs because of data privacy restrictions. We examined all deaths of the included patients captured within the TriNetX network; however, deaths outside of the participating HCOs are not well captured. It should also be noted that we had not access to time in therapeutic range of International Normalized Ratio (INR) for VKA-treated patients, so there are uncertainties about the quality of anticoagulation therapy in these patients. Finally, our main objective was to investigate the association of prior VKA or DOAC therapy with short-term prognosis after outpatient COVID-19 diagnosis. For this reason we did not take into account the anticoagulation therapy once patients were diagnosed of COVID-19, since our interest was on the previous use of VKAs or DOACs. For the same reason, we have not analyzed the potential role of other pharmacological therapies such as antidiabetics agents or insulin, although we recognize influence of such therapies on outcomes.In COVID-19 outpatients with cardiometabolic diseases, prior use of DOAC therapy compared to VKA therapy at the time of COVID-19 diagnosis might reduce the risk of composite arterial or venous thrombotic outcomes, without increasing the risk of bleeding.Additional file 1: Table S1. ICD-10-CM codes and LOINCs for the identification of COVID-19. Table S2. Clinical outcomes and ICD-10-CM codes.


Rapid adoption of robotic-assisted general surgery procedures, particularly for cholecystectomy, continues while questions remain about its benefits and utility. The objective of this study was to compare the clinical effectiveness of robot-assisted cholecystectomy for benign gallbladder disease as compared with the laparoscopic approach.A literature search was performed from January 2010 to March 2020, and a narrative analysis was performed as studies were heterogeneous.p < 0.05; 15 studies showed no difference; two studies showed shorter laparoscopic times), while conversion rates and intraoperative complications were not different. No differences were detected for the length of stay, surgical site infection, or readmissions. Across studies comparing single-port robot-assisted to multi-port laparoscopic cholecystectomy, there was a higher rate of incisional hernia; however, no differences were noted when comparing single-port robot-assisted to single-port laparoscopic cholecystectomy.Of 887 articles screened, 44 met the inclusion criteria . Four were randomized controlled trials, and four used propensity-matching. There were variable comparisons between operative techniques with only 19 out of 44 studies comparing techniques using the same number of ports. Operating room time was longer for the robot-assisted technique in the majority of studies (range 11–55 min for 22 studies, Clinical outcomes were similar for benign, elective gallbladder disease for robot-assisted compared with laparoscopic cholecystectomy. Overall, the rates of complications were low. More high-quality studies are needed as the robot-assisted technique expands to more complex gallbladder disease, where its utility may prove increasingly beneficial.CRD42020156945PROSPERO The online version contains supplementary material available at 10.1186/s13643-021-01673-x. Since the introduction of robot-assisted laparoscopic surgery, it has transformed numerous operations across specialties. In fact, roughly 3000 robotic units have been introduced into the United States of America (USA) over the last decade . The plaIn general surgery, the number of robot-assisted procedures has grown. Between 2012 and 2018, a large statewide collaborative described a more-than-10-fold increase in robot-assisted general surgery cases . Over onThis systematic review analyzes the clinical effectiveness of robot-assisted surgery compared with the laparoscopic approach for cholecystectomy for benign gallbladder disease.This review is part of a larger review commissioned by the Department of Veterans Affairs (VA) on the clinical outcomes and cost-effectiveness of robot-assisted procedures for general surgery. The review process was supported by a Technical Expert Panel (TEP) consisting of general surgeons, who specialize in robot-assisted surgery and are policymakers from across the country. This systematic review is reported using PRISMA standards, and the protocol for the larger review was registered in PROSPERO: CRD42020156945.All searches included PubMed, Embase, and Cochrane  from January 2010 to March 2020. The search used a broad set of common terms relating to “robotic surgical procedures” or “robotic-assisted,” “cost-effectiveness,” and “cholecystectomy.” We excluded studies published prior to 2010 since robot-assisted procedures were not widely being performed, and earlier studies likely captured surgeons within their “learning curve.” This decision was supported by our TEP  studied patients undergoing elective cholecystectomy for non-cancer indications, (2) included one group of patients treated with a robot-assisted technique, (3) had a comparison to patients treated with a laparoscopic approach, and (4) measured intraoperative, perioperative, or postoperative outcomes. Both randomized controlled trials (RCTs) and observational studies were included. Abstracts were included in the review and underwent the same quality assessment and duplication exclusion as full texts. All exclusion criteria are included in our literature flow  , 11. DatRCTs were assessed for quality (risk of bias) with the Cochrane Risk of Bias tool . We usedDue to the heterogeneity in clinical outcomes of both the RCTs and the observational studies, we did not conduct a meta-analysis. The data synthesis is narrative. We presented the data by grouping studies based on the number of surgical access ports used. This grouping is important given that clinical outcomes  may differ based on the number of ports, and interpretation of the data must include this context. The three comparison groups were as follows: robot-assisted multi-port compared with laparoscopic multi-port, robot-assisted single-port compared with laparoscopic multi-port, and robot-assisted compared with laparoscopic (unknown port number). Statistical analysis was done using R v4.0.2.The search identified 887 publications , and 44 were ultimately included in our study ; two studies followed two institutions, and the final seven were multi-institution but did not specify how many. Thirty-one studies were retrospective, and five were prospective.The risk of bias for RCTs was judged to have a low-to-moderate risk of bias with some aspects deemed as unknown . Over half of the other observational studies also showed that the robot-assisted approach took longer  found no significant difference between techniques Table  27, 38,,38, 46; The short-term outcomes reviewed were length of stay (LOS), surgical site infection (SSI), readmissions, and pain. Of the three RCTs that reported on LOS, only one demonstrated a shorter stay for the robot-assisted cholecystectomy (see Table p < 0.05) [None of the three RCTs that reported SSI rates demonstrated a statistically significant difference Table , and non < 0.05) . Of the  < 0.05) , 44, 51 Fifteen studies examined pain as an outcome, one RCT  and one p < 0.05). All studies that examined techniques with the same number of port sites showed no significant difference in hernia rates [The main long-term outcome of interest assessed was the rate of incisional hernias. Only 12 studies reported incisional hernia rates: two were RCTs , 46, andia rates , 28, 56.The certainty of evidence that OR time was longer for the robotic-assisted technique and that there was no evidence of differences in intraoperative complications, length of stay, or SSI rates was moderate due to imprecision in the evidence for these outcomes. The certainty of evidence for no difference in conversion rates was deemed high based on RCT data. We judged the certainty of evidence for no differences in readmissions and greater incisional hernia rates, when comparing single-port robot to multi-port laparoscopic techniques, to be low due to imprecision and inconsistency. Supplemental Data Content Our review found that OR time is longer for robot-assisted cholecystectomy as compared with laparoscopic cholecystectomy. There was no evidence of differences in intraoperative complications or conversion rates between surgical approaches. LOS, readmissions, and SSIs also had no evidence to support differences between techniques. Pain was examined, but the methods used within the studies were too heterogeneous to make conclusions regarding this outcome. Finally, rates of incisional hernias may be different when comparing approaches with different numbers of ports; however, when accounting for the use of the same number of ports, there was no evidence of a difference in outcomes.Our search yielded four RCTs, four propensity-matched studies, and 36 observational studies, and thus, this review on robot-assisted cholecystectomy is the largest to date  at 38 min, such a difference may represent a variety of modifiable factors such as surgeon learning curve, OR staff efficiency, and case selection . DiffereOur review had several limitations. The first is that the studies only examined surgery for benign, elective gallbladder disease—a process that is associated with low complication rates overall. Thus, differences in technique are unlikely to greatly affect these outcomes. As the use of the robot-assisted technique continues to expand, it is increasingly being applied in non-benign and non-elective settings for complex gallbladder disease and cases. Given the differences in complexity for such indications, the results from this review may not be generalizable to these populations. Second, we were unable to test for publication bias and cannot make any conclusions about its possible existence. Third, the quality of studies and heterogeneity in outcome measurement limited our conclusions. Fourth, this review did not address the differences in cost which may represent an important difference between techniques.In summary, OR time was found to be significantly longer for the robot-assisted technique, although a variety of factors may explain such differences. The other clinical outcomes did not differ between techniques for benign, elective gallbladder disease. Understanding the differences in outcomes for robot-assisted surgery is critical as the use of this technology is being introduced across surgical disciplines and will increasingly be used to address more challenging pathology. Future work should focus on RCTs or propensity-matched studies that include clinical endpoints as primary outcomes  and make appropriate comparisons when examining the number and type of ports used. Given the expanding use of robot-assisted cholecystectomy, these studies should also consider and control for other indications . An analysis of the costs of the robot-assisted technique relative to the potential benefits including an analysis of how the robot may improve or add new challenges for surgeon ergonomics will also contribute to the data used when considering the use of one platform over the other. Robotic technology will become more ubiquitous, thus understanding its impact remains of paramount importance in the quality control and implementation.Additional file 1: Supplemental Data Content 1. Search Strategies and Literature FlowAdditional file 2: Supplemental Data Content 2. Evidence tablesAdditional file 3: Supplemental Data Content 3. Heterogeneity of Pain Measurement Tools Used and Findings Comparing Robot-Assisted versus Laparoscopic CholecystectomyAdditional file 4: Supplemental Data Content 4. Quality assessment for Included RCT Studies (cochrane risk of bias tool)Additional file 5: Supplemental Data Content 5. Quality assessment for Included Observational Studies (ROBINS-i)Additional file 6: Supplemental Data Content 6. Certainty of Evidence for Cholecystectomy StudiesAdditional file 7: Supplemental Data Content 7. Characteristics and Results of Prior Systematic Reviews Examining Robot-Assisted versus Laparoscopic Cholecystectomy Outcomes


Athalia rosae, exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. The doublesex (dsx) gene is a well-conserved transcription factor that regulates sexual differentiation in insects. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. As a result, knockdown of Ardsx in haploid males caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. The same knockdown approach using diploid males caused complete male-to-female sex reversal; they were able to produce fertile eggs and exhibited female behaviors. The same RNAi treatment did not affect female differentiation. These results demonstrated that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.The sawfly, doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.The  Sex determination and sexual differentiation are controlled by a genetic cascade consisting of several sex-determining genes. Only the final element of the cascade is well-conserved, whereas upstream genes are highly evolutionary labile . Studies species ,3. The d insects ,5.Drosophila melanogaster, the primary dsx transcript undergoes sex-specific alternative splicing to produce a male-specific (DSXM) or a female-specific (DSXF) protein isoform [dsx in D. magna is expressed predominantly in males and is only required for male development [In  isoform . The DSX isoform ,8,9. Unlelopment ,5.dsx causes defects in male and female development, resulting in intersexuality in both sexes. Functional disruption of dsx causes the intersexual phenotype in several insect species. In the fruit fly Drosophila melanogaster, dsx-mutant females exhibit a male-specific pattern of abdominal pigmentation and develop a sex comb, which is a male-specific organ, whereas male-specific abdominal pigmentation and sex-comb formation are repressed in dsx-mutant males [dsx-mutant female silkworm, Bombyx mori, has a clasper-like structure and eight abdominal segments, both of which are male-specific traits, whereas the dsx-mutant male develops female-specific genital papillae [Trypoxylus dichotomus, which is famous for its large horns (on the head and thorax) in males, RNA interference (RNAi)-mediated dsx knockdown causes atrophy of the head horn and loss of the thoracic horn. On the other hand, dsx knockdown in females results in the appearance of a head horn [dsx dysfunction characterized so far are all partial; thus, functional disruption of dsx leads to an intersexual phenotype in most cases [dsx produces male- and female-specific isoforms, each of which is involved in male and female development.As the name implies, depleting nt males . The dsxpapillae ,12. In tead horn . Howeverst cases ,18,19,20Athalia rosae (Hymenoptera: Tenthredinidae), belongs to the Symphyta, which is the most primitive infraorder in the Hymenoptera. Similar to other hymenopteran insects, the sexual fate of the sawfly is determined by the single-locus complementary sex determination (CSD) system, in which heterozygosity at a single locus (the CSD locus) determines femaleness in diploid individuals, whereas haploid individuals are hemizygous for the CSD locus and thus develop into males [dsx ortholog in the sawfly and designated the gene Ardsx [Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner, yielding female- and male-specific isoforms  at almost all developmental stages. Our previous findings demonstrate that ArdsxM is required for male development [dsx orthologs so far identified in other holometabolous insects, Ardsx is likely to act at the bottom of the sex determination cascade and regulate sexual development in the sawfly. Notably, unlike other holometabolous insects, knockdown of Ardsx during the pupal stage in males caused almost complete male-to-female sex reversal in the external genitalia, whereas the same knockdown in females had no effect on sexual phenotype. However, the effect of Ardsx knockdown on male differentiation of internal genitalia, including the gonads, was extremely weak. This may be because the effect of Ardsx knockdown is transient—Ardsx expression was transiently knocked down from the last-instar larval stage to the adult stage in the previous study. It remains unclear whether Ardsx knockdown causes total male-to-female transformation and is dispensable for female development.The sawfly, to males ,22. In ane Ardsx . Ardsx pelopment . Thus, lArdsx expression during the entire lifespan, including the embryonic stage when Ardsx initiates sex-dimorphic expression [Ardsx were given to females after parental RNAi in order to extend the duration of RNAi-mediated knockdown of Ardsx expression for as long as possible. This RNAi treatment caused male-to-female sex reversal, not only in the external genitalia, but also in the gonads: the RNAi-treated males had ovaries containing fertile eggs. On the other hand, females that received the same RNAi treatment exhibited a normal phenotype, supporting our previous finding that Ardsx is dispensable for female differentiation. Thus, unlike in other insect species, functional depletion of dsx in the sawfly did not cause an intersexual phenotype but induced total male-to-female transformation. Some evolutionary implications of these findings will be discussed later.To address this question, it is important to knock down pression . In the pression . In the A. rosae) were reared continuously at 25 °C under 16-h-light/8-h-dark conditions and fed fresh Japanese radish leaves  cultivated as described previously [yellow fat body (yfb) mutant strain was also used in the present study. The wild-type fat body is greenish blue in color but animals homozygous for yfb have fat bodies that are yellow [yfb display intermediate coloration (yfb (yfb/yfb) was mated with a wild-type haploid male, and their progeny were subjected to sib-crossing, where a single diploid female (+/yfb) was crossed with a single haploid male (+ or yfb). Such interbreeding was repeated multiple times. The resulting males heterozygous for yfb (+/yfb) were treated as diploids. To discriminate diploid males from females, molecular sexing of each animal was determined according to the expression pattern of Ardsx, investigated by RT-PCR, as described below, at more than 7 days after emergence, when the effect of Ardsx knockdown had disappeared. Individuals that preferentially expressed female-type Ardsx (ArdsxF) were recognized as females, whereas individuals that predominantly expressed male-type Ardsx (ArdsxM) were defined as males  as an internal standard for the RT-PCR [Extraction of total RNA was performed using ISOGEN , according to a previously described protocol . The RT-e RT-PCR . The priArdsx  were the same as those used in our previous study [EF-1 alpha gene as an internal standard. The threshold cycle (CT) value was normalized with the CT value of the EF-1 alpha gene using Multiple RQ software . We confirmed that the above primer sets do not produce off-target products by checking dissociation curves of the qPCR products. The relative value of Ardsx expression against EF-1 alpha expression was obtained in quadruplicate and the ratio of Ardsx expression calculated.Quantitative RT-PCR assays were performed according to a previously described protocol . The prius study . The ArEArdsx isoforms were amplified with the primer pairs ArdsxdsRNAF1-ArdsxdsRNAR1 and ArdsxdsRNAF2-ArdsxdsRNAR2, as described previously [Two sequences shared among the male and female eviously , and sereviously .Ardsx expression in order to prolong the duration of RNAi-mediated mRNA knockdown, further injections of dsRNA into progeny obtained via parental RNAi were performed at the second, third, and final instar larval stages. When the larvae were subjected to RNAi, dsRNAs were injected into the dorsal hemocoel in the second abdominal segment of a larvae. Approximately 1 μL of dsRNA solution with a concentration of 100 ng dsRNA/μL was injected into each animal.The dsRNAs were injected into larvae and pupae according to a previously described protocol . ParentaCuticle specimens of external genitalia were prepared as described previously . The speArdsx-knockdown males were used at least 14 days after eclosion, as we discovered that egg maturation in Ardsx-knockdown males took longer than in wild-type females. Naïve males, which had never met a female and had never experienced mating, and virgin females were isolated in vials before use. To examine whether Ardsx-knockdown males exhibited courtship behaviors, two wild-type virgin females were introduced into a vial  with two Ardsx-knockdown males. To investigate whether Ardsx-knockdown males were able to attract naïve wild-type males, a single Ardsx-knockdown male was introduced into a vial with a naïve wild-type male. Video recording  started immediately after introducing the virgin females . The typical courtship behavior of the sawfly based on previous reports [Courtship index: the fraction of time spent in any aspect of courtship behavior, including chasing, touching, licking, and attempted copulation within the first 10 min of the assay. Copulation latency: the time from presentation to copulation. Copulation success: percentage of males achieving copulation within the first hour of the assay.All behavioral experiments were carried out near a window with sunlight at 25 °C. Males and virgin females were typically used at 7 days post-eclosion.  reports ,28,29 ishttps://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/download.html, accessed on 6 September 2021). The Shapiro–Wilk test was used to evaluate whether the data obtained by our experiments showed a normal distribution. Since the number of samples was less than 25 and did not show a normal distribution, the significant difference test between the two groups was performed using the Mann–Whitney U test. The data obtained in this study were displayed in box plot format. The box plot was created using EZR software with default settings.All statistical processing was performed using Easy R (EZR) software  is more efficient than embryonic injection in terms of penetrance of the effect [Ardsx were given to females produced after parental RNAi in order to extend duration of RNAi-mediated knockdown of Ardsx expression for as long as possible  does not occur during spermatogenesis in the male sawfly  because Ardsx knockdown of diploid males caused male-to-female sex reversal as observed in both the external genitalia and gonads  . In insects, dsx is characteristic in that it produces different isoforms in males and females as a result of alternative splicing, and each isoform is responsible for male and female sexual development [Daphnia magna and D. pulex, dsx orthologs are essential for male development and do not exhibit sex-specific splicing , belongs to the Symphyta infraorder, which is the most primitive infraorder in the Hymenoptera. Thus, it is quite reasonable to speculate that dsx in the sawfly is only required for male development and is dispensable for female development , oogenesis occurs even if the sex of the surrounding somatic cells is male. DSX is dispensable for oogenesis, and factors that are expressed only in XX germ cells, such as a protein product of ovarian tumor (otu), as well as the germline-specific isoforms of OVO and SXL, are essential for oogenesis [D. melanogaster, in that DSX is not required for oogenesis, because Ardsx-knockdown individuals produce normal eggs  plays a pivotal role in the development of the male-specific nervous system that is required for male sexual behavior [fru transcripts (Mfru) cause defects in male courtship behavior and sexual identity [Musca domestica, the mosquitoes Anopheles gambieae and Aedes aegypti, the parasitic wasp Nasonia vitripennis, and several hemimetabolous insects, including the desert locust Schistocerca gregaria and the German cockroach B. germanica, suggest a conserved evolutionary role of the Fru protein in male courtship behavior [fru, not dsx, plays an essential role in the development of male-specific neural networks in the sawfly.Despite behavior . In D. mbehavior ,48,49. Midentity ,52,53,54behavior ,58,59,60A. rosae, the courtship index for Ardsx-knockdown males was significantly lower than that for wild-type males  is not necessary for most aspects of male courtship behavior, but it is required for the male courtship song [Mfru and dsxM functions to accomplish normal male development [dsxM acts together with Mfru in the differentiation of male-specific neurons in the abdominal ganglion [dsx in Drosophila, Ardsx has a partial but important role in specifying all aspects of male courtship behavior in the sawfly.However, in pe males B. This inogaster ,62. In Dhip song ,61. DSXMhip song . Severalelopment . Also, dganglion . SimilarArdsx-knockdown males did not attract wild-type males  occurs only in fertilized eggs, where maternal and paternal genomes coexist [Nvtra allele is prevented by maternal imprinting [Interestingly, when haploid males were subjected to repeated injections of Ardsx1 dsRNA, the resulting pe males D–F. Thesid males J. How don system ,66. In N coexist . Unfertiprinting . It is pD. melanogaster, the dsx gene acts at the bottom of the sex-determination cascade that induces appropriate sexual differentiation in each sex according to upstream genetic sex-determining signals [dsx leads to the intersexual phenotype in both sexes [B. germanica, where knockdown of dsx causes partial male-to-female sex reversal but has no effect on female development [dsx by repeated injections of dsRNA in the sawfly induced complete male-to-female transformation. The resulting pseudofemales successfully copulated with wild-type males and produced fertile eggs. Remarkably, the total male-to-female transformation was observed only when diploid males were subjected to the RNAi treatment. A caveat is that we were not able to confirm whether Ardsx expression in the examined diploid males was indeed knocked down during development from the embryonic to pupal stages by the RNAi, as their genetic sex can only be identified by RT-PCR analysis performed at least seven days after emergence (see Ardsx was significantly reduced in diploid females (Ardsx expression in diploid males as well. These results suggest that diploidy is also important for normal female development. The sexual fate of the sawfly is determined by the CSD system, in which heterozygosity at the CSD locus determines femaleness in diploid individuals, whereas haploid or diploid individuals hemizygous or homozygous for the CSD locus develop into males [Ardsx; thus, they do not exhibit femaleness even though they have diploid genomes. Further studies are needed to validate this hypothesis.In  signals ,3. Thus,th sexes ,18,19,20elopment . Distincence see . However females C, we expto males ,22. At f


Radiotherapy is one of the most important treatment options against cancer. Irradiation of cancerous tissue either directly destroys the cancer cells or damages them such that they cannot reproduce. One side-effect of radiotherapy is that tumor-surrounding normal tissue is inevitably also irradiated, albeit at a lower dose. The resulting long-term damage can significantly affect cognitive performance and quality of life. Many studies investigated the effect of irradiation on normal-appearing brain tissues and some of these correlated imaging findings with functional outcome. This article provides an overview of the examination of radiation-induced injuries using conventional and enhanced MRI methods and summarizes conclusions about the underlying tissue changes. Radiation-induced morphologic, microstructural, vascular, and metabolic tissue changes have been observed, in which the effect of irradiation was evident in terms of decreased perfusion and neuronal health as well as increased diffusion and atrophy.Radiotherapy is part of the standard treatment of most primary brain tumors. Large clinical target volumes and physical characteristics of photon beams inevitably lead to irradiation of surrounding normal brain tissue. This can cause radiation-induced brain injury. In particular, late brain injury, such as cognitive dysfunction, is often irreversible and progressive over time, resulting in a significant reduction in quality of life. Since 50% of patients have survival times greater than six months, radiation-induced side effects become more relevant and need to be balanced against radiation treatment given with curative intent. To develop adequate treatment and prevention strategies, the underlying cause of radiation-induced side-effects needs to be understood. This paper provides an overview of radiation-induced changes observed in normal-appearing brains measured with conventional and advanced MRI techniques and summarizes the current findings and conclusions. Brain atrophy was observed with anatomical MRI. Changes in tissue microstructure were seen on diffusion imaging. Vascular changes were examined with perfusion-weighted imaging and susceptibility-weighted imaging. MR spectroscopy revealed decreasing N-acetyl aspartate, indicating decreased neuronal health or neuronal loss. Based on these findings, multicenter prospective studies incorporating advanced MR techniques as well as neurocognitive function tests should be designed in order to gain more evidence on radiation-induced sequelae. Radiotherapy (RT) is an indispensable part of the treatment of the majority of primary and metastatic brain tumors ,2,3,4. SQuantitative volumetric MRI measurements have been used to observe radiation-induced anatomical differences. Several studies focused on volumetric changes of the whole brain (WB), white matter (WM), and grey matter (GM) ,35,36,37Petr et al.  examinedSeveral studies of the St Jude Children’s Research Hospital in Memphis focused on volume loss of normal-appearing brain tissue of medulloblastoma patients who received radiotherapy during childhood ,32,33,34Liu et al.  found coSeveral studies focus on radiation-induced volumetric changes in the hippocampus. Takeshita et al.  detectedChanges in the amygdala were focused on by Huyngh-Le et al. . In a coAnother study evaluated the effect of region-specific sparing in the cerebral cortex . The autTwo studies focused on volume changes of the cerebellum after RT ,52. RascDiffusion imaging allows the assessment of changes in the microstructure of brain tissue in vivo. Whereas diffusion-weighted imaging (DWI) provides basic information on the apparent diffusion coefficient (ADC), also referred to as mean diffusivity (MD), diffusion-tensor imaging (DTI) provides additional information about fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivity (RD). Several studies focused on microstructural tissue changes in white matter structures after radiotherapy. The most prominent effects are decreasing FA ,79,80,81Nagesh et al.  measuredq did not change. Stable values in proton density indicated that resolving oedema was not a likely cause for the decrease in diffusion. Likewise, quantitative T1 values did not change significantly; thus, changes in myelin are also an unlikely cause. A possible explanation for decreasing diffusion would be axonal swelling, similar to acute stroke [Contrary to commonly found results, the recent study by Raschke et al.  reportede stroke , only ate stroke  also suge stroke . In a foe stroke  could ree stroke  in severe stroke  and Dünge stroke . Zhu et e stroke  also foue stroke , suggestb-values, which shows that MD, AD, and RD increased stronger for lower b-values, while the magnitude of FA decrease was greater for high b-values. From the stronger alterations for lower b-values, the authors suggested that the biological changes are prominently in the extracellular environment and are possibly caused by increased vascular permeability and neuroinflammation. Consequently, they recommended the future investigation of extracellular diffusion by measurements with different b-values. Dose-dependent normal-appearing WM changes were also identified by Hope et al. [Connor et al.  investige et al. , where Me et al.  reportede et al.  measurede et al.  reportede et al. ,87 did nConnor and colleagues  evaluatetrans (volume transfer constant between blood plasma and extravascular extracellular space (EES)), ve (volume of EES) and vp  [Radiation-induced vascular changes can be detected with perfusion-weighted imaging (PWI) methods such as dynamic susceptibility contrast (DSC), dynamic contrast enhancement (DCE), and arterial spin labeling (ASL). Most PWI studies found a perfusion decrease after radiotherapy, characterized by decreasing cerebral blood volume (CBV) ,99,100,  volume) ,103. TabUsing DSC, Wenz et al.  found a Contralateral GM perfusion changes using ASL were assessed by Petr et al.  in a cohAs opposed to the results of most studies, Jakubovic et al.  reportedtrans and ve in WM and GM after irradiation in 17 glioblastoma patients over a period of up to 185.7 days. While global Ktrans in WM and GM did not change significantly, ve showed a significant increase in GM 101.6 days after RT. For both parameters, no dependency on the radiation dose was found. The authors interpreted the increase in ve as an indicator for decreasing cell density after RT. Cao et al. [trans and vp to be significantly increased during and after RT in the cerebral tissue of 10 patients with low-grade gliomas, meningioma, cranopharyngeoma, and benign tumors. vp showed a significant increase of 11% at week six during RT in high-dose areas > 40 Gy and a decrease after RT. In intermediate dose volumes of 20 to 40 Gy, the maximal increase was measured one month after RT, also followed by a decrease six months after RT. The authors interpreted the initial increase in vp with fast vessel dilation during radiation [trans increased significantly by 39% in medium-dose regions and 52% in high-dose regions in week six during RT. In both dose regions, Ktrans recovered to pre-RT values six months after RT. Both parameter alterations correlated with the dose at the times of scans until one month after irradiation.Using DCE, Fahlström et al.  analyzedo et al.  calculatadiation ,105,106 adiation ,105,107.Petr et al.  comparedFew studies have assessed radiation-induced microvascular changes by visualizing microbleeds with susceptibility-weighted imaging (SWI). In a longitudinal study over 20 years, Lupo et al.  detectedMagnetic resonance spectroscopy (MRS) allows for non-invasive detection of metabolic changes in the brain, and thus holds potential to quantify radiation-induced neurotoxicity. The metabolites N-acetyl aspartate (NAA), a marker for neuronal health and density; choline (Cho), a marker of membrane integrity and proliferation; and creatine (Cr), a bioenergetic metabolite ,112, havKaminaga et al.  reportedEarly radiation-induced metabolic changes were observed by Movsas et al.  three toIn several studies, a recovery of the metabolic alteration was reported. Esteve and colleagues  investigDose-dependent metabolic changes and recovery in the first six months following radiation were detected by Lee et al.  in normaContrary to the previous results, Sundgren et al.  measuredA dose-dependent NAA/Cr decrease was reported by Rutkowski et al.  in 43 gln = 10) and phosphorus (n = 13) MRS were evaluated in a cohort of 23 patients, mainly malignant gliomas, by Szigety and co-workers [Proton (-workers . The subIn addition to studies of acute and early delayed effects, other studies considered the long-term damage of radiation ,125,126.Waldrop et al.  focused We summarized the current status of advanced MR imaging techniques for the assessment of the radiation-induced anatomical, morphological, and metabolic damage of normal-appearing brain tissue in primary brain tumor patients. Advanced MRI techniques offer the opportunity to visualize tissue changes not detectable with common T1- and T2-weighted MRI and highlight early tissue alterations. Anatomical and morphological damage was indicated by atrophy measured in both grey and white matter as well as in several substructures. Various studies reported on volume decrease after irradiation, which was more pronounced than that observed in normal aging or in patients with Alzheimer’s disease. Additionally, natural maturation of the brain in pediatric patients was hampered following irradiation. The effect of irradiation on the tissue microstructure was assessed using diffusion-weighted and diffusion-tensor imaging and revealed changes indicative of demyelination, axonal loss, and transient cerebral oedema. Perfusion-weighted imaging showed decreased cerebral blood flow and cerebral blood volume. Finally, metabolic changes assessed by MR spectroscopy indicated adverse effects on neuronal health, the cell membrane, and myelin.Based on the reviewed results, future studies of normal tissue examination after irradiation may incorporate a few improvements. First, when assessing radiation damage to the brain, increased attention should be paid to the impact on the patient’s neurocognitive functioning and quality of life by implementing adequate tests and correlating results with imaging findings. Second, future studies may benefit from evaluating multiple MR contrasts in the same patient cohort, since the combination of diverse information allows more detailed conclusions on the underlying tissue alterations. Third, to improve future publications on irradiation-induced MR changes and their inter-comparability, we strongly suggest a more comprehensive description of the used methodology. In particular, it is necessary to clearly describe the exclusion criteria for abnormal tissue, especially whether and how abnormal tissue was censored at various timepoints. Fourth, multicenter studies including larger numbers of patients and obtaining data on different scanners will increase the clinical relevance of the received results. Finally, to avoid the influence of infiltrating tumor cells on the surrounding normal-appearing brain, cohorts of patients with benign brain tumors or preferably head–neck cancers may be assessed, since tissue changes of the brain can be assumed to solely stem from irradiation.In the era of highly conformal photon beam techniques, as well as the worldwide increasing availability of proton and particle beams, advanced MRI may serve as an objective measure for the selection of brain tumor patients. This review shows the potential of MRI for normal tissue response assessment, and thus, it should be included in future prospective clinical trials.The literature search was based primarily on PUBMED combined with the consideration of secondary literature of the studied publications and google inquiries. The development of the literature collection was completed by December 2020. The data is based on research papers, while review articles were studied and consulted for further publications. Special attention by collecting publications was paid to the exclusion of abnormal tissue and the focus on normal-appearing tissue. The following shows a partial selection of the keywords used: Radiation-induced changes/injuries/alterations in brain/CNS; early and late/longitudinal radiation effects; response to radiotherapy/irradiation; cognitive impairment; normal-appearing brain tissue; conventional MRI; functional/quantitative MRI; microstructural/vascular/anatomical/morphological/metabolically changes in normal brain; Whole brain/white matter/grey matter volume change/loss/decrease; atrophy; diffusion tensor/weighted imaging; microstructure; diffusivity; demyelination; WM damage; perfusion-weighted imaging; susceptibility-weighted imaging; dynamic susceptibility contrast; dynamic contrast enhancement; arterial spin labeling; relative/absolute blood flow/volume; intra and extracellular space transition; histopathological changes; vessel dilatation; permeability; microbleeds; SWI lesion; MR spectroscopy; metabolism; NAA, neuronal marker; choline, cell membrane synthesis; creatine, energy metabolism; and several more. MR technique-specific keywords were combined with tissue alterations due to irradiation damage and the requirement for normal tissue observation with AND/OR research functions on PUBMED. Abnormal tissue changes due to irradiation were partially excluded.


The purpose of this study was to explore the relationship between parenting styles and career decision-making difficulties in college students, and uncovered the mediating roles of core self-evaluation and career calling. A total of 1,127 undergraduates were recruited to complete the questionnaires about parenting styles, core self-evaluation, career calling, and career decision-making difficulties. The results showed that: (1) Positive and negative parenting styles could positively predict career decision-making difficulties in college students. (2) Core self-evaluation and career calling mediated the relationship between parenting styles and career decision-making difficulties. Sequential dual mediators only found in which positive paternal and maternal parenting styles predict career decision-making difficulties through core self-evaluation and career calling. (3) Further analysis revealed gender difference in the relationship between parenting styles and career decision-making difficulties. The relation between paternal positive parenting style and career decision-making difficulties was significant in male students, but absent in female students; the relation between maternal positive parenting and career decision-making difficulties and the relation between paternal negative parenting and career calling were significant in female students, but absent in male students; and the relation between career calling and career decision-making difficulties was greater in male than in female. The current study expanded and deepened those existing understandings about the relationship between parenting styles and adolescents’ career decisions, so as to further reveal its internal mechanism and provide more reasonable suggestions and targeted guidance for career counseling. Recently, the employment pressure is increasingly severe under the situation of saturated job market . Until nResearches showed that individuals’ career decision-making was closely related to environmental factors, such as family e.g.,  and sociAlthough these findings consistently showed that parenting styles are an effective predictor of career decision-making difficulties, there is still a lack of research on the mechanism underlying this relationship. Thus, it is imperative to expand the previous research and examine the intermediate variables  between parenting styles and career decision-making difficulties among college students. Previous studies showed that the predictive effect of parenting styles on career decision-making was influenced by some internal factors, such as core self-evaluation e.g., , self-efCore self-evaluation is one of the most important personality factors. Core self-evaluation, as a cognitive factor, refers to an individual’s most basic evaluation of his or her own abilities and values . As a tyFollowing core self-evaluation, career calling could act as the second mediator proximal to career decision-making difficulties. Career calling is a dynamic process that takes on different states  as life experiences change . SearchiIndividuals with a high sense of career calling hope that their work can make a valuable contribution to society, and experience inner pleasure and self-realization in their work . Career There was obvious gender difference, however, in career decision-making difficulties. Specifically, Researches have showed that gender differences emerged in emotional and personality-related factors in career decisions, not in cognitive factors e.g., . Male paThe current study examined the important prediction of parenting styles for distal career decision-making difficulties in college students, and further revealed the internal mechanism. The study hypothesized a sequential dual mediator model in which core self-evaluation and career calling sequentially function as mediators in the prediction of parenting styles for career decision-making difficulties. Specifically, this study hypothesized that: (1) positive/negative parenting styles could negatively/positively predict career decision-making difficulties; (2) parenting styles could also influence career decision-making difficulties through core self-evaluation and career calling; (3) gender differences existed in the process of parenting styles to predict career decision-making difficulties through core self-evaluation and career calling. It will help researchers to understand the internal and external causes of career decision-making difficulties for college students.A total of 1,240 juniors and seniors were recruited from five public universities in Jinan, China. The questionnaires were released on the Internet, which was freely filled in by qualified subjects with the consent of the teacher and the students. 113 participants were excluded due to responding sloppily or in sequence. The effective rate of date analyzed was 90.89%. Of the sample, this study included 47.2% male, and 59.6% were junior. All of the students received a pen as compensation for their participation.Short-Egna Minnenav Barndoms Uppfostran for Chinese (s-EMBU-C) , was adaThe Core Self-Evaluation Scale  was usedThe Calling Scale  was usedCareer decision-making difficulties were measured by the Career Decision-Making Difficulties Questionnaire . There aT-test was performed to explore gender differences. In order to further determine gender differences in the mediating effect of career calling and core self-evaluation on parenting styles and career decision-making difficulties, multi-group comparison analysis was performed.SPSS 22.0 and AMOS 24.0 were used to analyze the data, including descriptive statistical analysis and correlation analysis. Structural equation modeling analysis was conducted separately for male and female using AMOS 24.0. In the unconstrained model, all parameters were allowed to vary across groups, whereas in the constrained model, all path coefficients were fixed as equal across groups. A better model fit in the unconstrained model indicates that the relations between parenting styles and career decision-making difficulties differ across the two groups.Model fit of all the structural equation models was assessed by the comparative fit index (CFI), the Tucker-Lewis Index (TLI), the root-mean-square error of approximation (RMSEA), and the chi-square statistic. Because the chi-square statistic is sensitive to large samples, model fit indices were used as the primary criteria to evaluate model fit. The TLI and the CFI range between 0 and 1, with values above 0.90 indicating adequate model fit . A rule ps > 0.05). In order to explore whether gender moderated the links between parenting styles and career decision-making difficulties, independent sample T-test and Pearson correlation analysis was conducted on the variables in male and female samples, respectively (see ps < 0.05) except that the paternal negative parenting style was uncorrelated with career decision-making difficulties  in male sample, the paternal and maternal parenting style were uncorrelated with career decision-making difficulties (ps > 0.05) in female sample.vely see . The res2/df = 7.04, RMSEA = 0.07, CFI = 0.95, TLI = 0.93).Based on previous studies and the analysis of the relationship among variables in this study, the mediating role of core self-evaluation and career calling in the relationship between parenting styles and career decision-making difficulties was investigated. A multi-mediation model was performed by taking the four dimensions of parenting styles as predictive variables, core self-evaluation and career calling as mediating variables, and career decision-making difficulties as outcome variables. The results showed that the model fitted well (χp < 0.01)and career decision-making difficulties , career calling positively predicted career decision-making difficulties  (see p < 0.001), career calling  and career decision-making difficulties , indicating that core self-evaluation and career calling played a mediating role between positive paternal parenting style and career decision-making difficulties. The indirect effect was 0.044, accounting for 38.94% of the total effect (see p < 0.01), it had no significant predictive effect with core self-evaluation and career calling (ps > 0.05); Positive maternal parenting style was not found to predict career decision-making difficulties (p > 0.05), but it was found to (positively) predict career calling , indicating that positive maternal parenting style indirectly affected career decision-making difficulties by college students’ career calling. The indirect effect was 0.051, accounting for 75% of the total effect (see ps > 0.05), but it was found to (positively) predict core self-evaluation , indicating that negative maternal parenting style indirectly affected career decision-making difficulties by college students’ core self-evaluation and career calling. The indirect effect was –0.017, accounting for 41.47% of the total effect  see . Positivfect see ; Althougfect see . Negativfect see . To sum 2/df = 3.851, RMSEA = 0.073, GFI = 0.943, NFI = 0.949; Female: χ2/df = 3.957, RMSEA = 0.071, GFI = 0.948, NFI = 0.929, see To further verify whether there were significant differences between male and female in the mediating model, we tested the structural equation model on two samples. The results showed that the model fit well for both male and female sample . It showed that the model is unstable in male and female samples. On the basis of M2, the structure weight of the variables was restricted to be equal (M3). The results showed that the fitting index of M3 and M2 existed a significant difference , suggested that there were considerable differences in this indirect effect model between male and female  under the condition that the measurement weights of both two groups of subjects were equal. Compared with the M1 without any restriction, the results revealed that the fitting degree of M2 was better , and a significant difference existed in M2 and M1 ; the relationship between paternal positive parenting style and career decision-making difficulties was significant in male students, but absent in female students ; the relationship between maternal positive parenting and career decision-making difficulties was significant in female students, but absent in male students ; and the relationship between paternal negative parenting and career calling was significant in female students, but absent in male students .Path coefficient comparison was further performed, and found that there were considerable differences between male and female students on the four various paths: Respectively, the relationship between career calling and career decision-making difficulties was greater in male than in female  significantly predicted career decision-making difficulties, which consistent with previous studies e.g., . This reMoreover, the result showed strong positive prediction function for negative paternal parenting style to career decision-making difficulties, which was contrary to previous research e.g., . This maThe current study was particularly interesting in illuminating potential predictors of core self-evaluation and career calling. As expected, it revealed that the role of parenting styles on career decision-making difficulties can be realized through the bridge of core self-evaluation and career calling. More specifically, positive paternal parenting style and negative maternal parenting style could not only directly predict career decision-making difficulties, but also indirectly predict career decision-making difficulties through core self-evaluation and career calling. The positive parenting style of mothers indirectly predicted career decision-making difficulties through career calling. These predictive effects were consistent with results of prior studies . ParentsThe results also supported social cognitive career theory , which hIt should be pointed out that the multiple mediators found it has certain gender differences in this study. This is mainly reflected in the following factors.Firstly, in the sample of male students, the degree of career calling predicts career decision-making difficulty is higher than that of female students. In the context of Eastern culture, researchers believe that career calling is more rooted in internal forces than driven by external forces . In addiSecondly, females who perceive their father as exhibiting a strict control and rejection over their behavior tended to report less career calling. This may be because female are more susceptible to external influences than male . And carThirdly, the relationship which positive paternal parenting style directly predicted career decision-making difficulties only exist in the sample of male, and the relationship which positive maternal parenting style directly predicted career decision-making difficulties only exist in the sample of female. In terms of positive parenting, “like-sex” plays an important role, that is, mother often participate more in the growth of their daughter, and son are often disciplined more by their father . CombineIn the present study, several limitations should be noted. First and foremost, the current study drew upon cross-sectional date to examine the dual mediator model. While the conceptual flow of this model was based on the temporal career decision-making difficulties, the temporal sequence and potential causality cannot be fully established without longitudinal date. To future consolidate the utility of parenting styles, it is necessary for future research to longitudinally validate the relationships in the current study. Then, as date in the current study was self-reported, some of the identified associations may be inflated. Future studies are warranted to employ multi-informant and multi-method designs to minimize shared informant. For example, parental styles can be reported by parents. Finally, the current study only examined three single pathways with gender differences. Future studies should further validate the differences between this dual mediating effect in the sample of male and female in order to better understand the potential role of parenting styles in predicting career decision-making difficulties.The conclusions can be drawn as follows: (1) positive and negative paternal parenting styles have a directly significant positive predictive effect on career decision-making difficulties in college students; (2) core self-evaluation and career calling play a chain-mediated role in the relationship that positive paternal parenting style predict career decision-making difficulties and negative maternal parenting style predict career decision-making difficulties; (3) gender differences exist in the process of parenting styles to predict career decision-making difficulties through core self-evaluation and career calling.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Shandong Normal University. The patients/participants provided their written informed consent to participate in this study.JS and DH conceived the idea and developed the materials. BH and HL carried out the data collection. XT and SX wrote and revised the manuscript. KA polished the language of this manuscript. JS, DH, BH, and HL provided critical feedback. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The three major conventional treatments: surgery, chemotherapy, and radiation therapy, have been commonly performed for lung cancer. However, lung cancer is still the leading cause of cancer-related mortality. Immunotherapy has recently emerged as a very effective new treatment modality, and there is now growing enthusiasm for cancer immunotherapy worldwide. However, the results of clinical studies using immunotherapy are not always favorable. Understanding the steps involved in the recognition and eradication of cancer cells by the immune system seems essential to understanding why past immunotherapies have failed and how current therapies can be optimally utilized. In addition, the combination of immunotherapies, such as cancer vaccines and immune checkpoint inhibitors, as well as the combination of these therapies with three conventional therapies, may pave the way for personalized immunotherapy. In this review, we summarize the results of immunotherapies used in phase III clinical trials, including immune checkpoint inhibitors, and discuss the future prospects of immunotherapies in lung cancer treatment. Lung cancer is one of the most deadly of solid cancers . AdvanceCurrently, there is no doubt that immune checkpoint inhibitors (ICI) are the mainstay of current immunotherapy in lung cancer. In particular, the results of the ICI clinical trials in 2015  were sensational ,7. BeforAntigen-specific vaccines are intended to induce specific anti-tumor immunity against specific tumor-associated antigens (TAAs). Conversely, tumor cell vaccines, consisting of autologous or allogeneic tumor cells, often expose the immune system to a variety of unknown tumor antigens . SeveralA phase III trial of MVP  with or without monthly mycobacterium vaccine administration (SRL172) for advanced NSCLC was performed and showed no survival benefit in 2004 . A phaseTen years later, the results of clinical trials using other cancer vaccines were also published. In 2014, a placebo-controlled phase III trial of a vaccine targeting a major related ganglioside (Racotumomab-Alun) as maintenance therapy for advanced NSCLC previously treated with platinum was conducted in Cuba and showed advantage in survival rate of both PFS and OS, although it was suggested that the low rate of subsequent therapy may have influenced the results . In addiPatients with NSCLC which express TAA melanoma-associated antigen-A3 (MAGE-A3) are often associated with poor prognosis. The MAGE-A3 vaccine is a combination of recombinant MAGE-A3 protein and the immunostimulant AS02B. It was evaluated in a large, double-blind, randomized phase III trial (MAGRIT) in 2015. This trial had negative results. There was no improvement in disease-free survival (DFS) in the vaccine group compared to placebo, either in the overall population or in patients who did not receive chemotherapy .Agonist antibody to costimulatory molecule or costimulatory receptor was clinically used in clinical trials as a promising immunotherapy. A phase III trial of cisplatin plus carboplatin plus paclitaxel in 2011  and gemcA phase III trial of cisplatin plus gemcitabine with or without low-dose IL-2 for advanced NSCLC did not show advantage in survival rate on 1-year survival rate in 2011 . Other rA meta-analysis of cancer vaccine therapy in NSCLC reported that both PFS and OS were prolonged, however, the stage of the patients was different. Therefore, the results of the analysis need to be understood with caution . ConsideImmune checkpoints are proteins on the surface of T-cells and other immune cells that function as negative regulators of immune activation by a variety of antigens, including tumor antigens. ICIs are a class of immunotherapeutic agents that harness the intrinsic immune response to tumor antigens by removing the brakes on T-cell activation by antigen-presenting cells (APCs). The first checkpoint discovered was cytotoxic T-lymphocyte antigen-4 (CTLA-4), and ipilimumab, an ICI developed to target CTLA-4, has shown prolonged survival in melanoma . Such ICAmong the various checkpoint pathways, the PD-1 pathway, consisting of the receptor (PD-1) and its reciprocal ligands ), CTLA-4 pathway, have been most intensely studied in NSCLC in recent years. Monoclonal antibodies targeting PD-1 , PD-L1 , or CTLA-4 (ipilimumab), have been investigated in clinical trials for lung cancer and have demonstrated significant improvements in survival.Clinically, nivolumab showed an OS advantage over docetaxel, the conventional standard of chemotherapy in 2015. Of note, there is the tail plateau effect seen in nivolumab-treated patients, in which the OS and PFS curves almost cease to decline after a certain point, indicating a long-term progression-free survival effect that was thought to be impossible to achieve with conventional therapy  ,7. The pPembrolizumab is the most widely used ICI in lung cancer currently. KEYNOTE-010 trial showed the efficacy of pembrolizumab for patients with previously treated, PD-L1-positive, advanced NSCLC . FurtherA randomized, double-blind, phase III trial of sintilimab (fully human anti-PD-1 antibody) with pemetrexed and platinum was conducted in China. In Chinese patients with previously untreated locally advanced or metastatic non-squamous NSCLC, the addition of sintilimab to pemetrexed and platinum-based chemotherapy significantly prolonged PFS with a manageable safety profile compared to chemotherapy alone (ORIENT-11) . In the In the first-line treatment of advanced NSCLC with PD-L1 expression rates of 50% or higher, cemiplimab monotherapy significantly improves OS and PFS compared with chemotherapy. As well as sintilimab, cemiplimab may provide a new treatment option for patients with high PD-L1-expressing NSCLC.The IMPower110 trial showed that atezolizumab alone significantly prolonged OS compared to platinum-based chemotherapy in patients with NSCLC of any histology with high expression of PD-L1 . In 2019The combination of atezolizumab with paclitaxel plus bevacizumab in chemotherapy-naive patients with metastatic non-squamous NSCLC patients showed its additional affect in IMPower150 study ,49. HoweAtezolizumab was shown to be effective and safe as neoadjuvant immunotherapy for stage IB-IIIB resectable NSCLC in the phase II trial (LCMC3) . In addiA phase III study comparing durvalumab and placebo in patients with non-resectable, locally advanced NSCLC who were progression-free after concurrent chemoradiation showed significant improvement in OS and PFS ,54.Based on the results of CheckMate 227, nivolumab plus ipilimumab is recommended as first-line therapy for NSCLC with metastatic disease, regardless of PD-L1 expression, although the amount of tumor mutation burden (TMB) may be related   was the first FDA-approved companion diagnostic test for ICI. IHC assays are performed using standard methods used in other IHC companion diagnostic tests, such as hormone receptor expression in breast cancer  and is r+ T-cell infiltration [In recent years, TMB has been paid attention to as another indicator of response to ICIs. The premise of this test is that as the number of non-synonymous mutations increases, a unique tumor neoantigen is generated that is recognized by the immune system and promotes tumor recognition and killing by adaptive immune cells when reactivated by ICI ,66. Thisltration ,71, as wltration , POLD1, ltration .In lung cancer, Rizvi and co-authors demonstrated TMB is a potential biomarker for immunotherapy . HoweverBecause many patients with advanced lung cancer are unable to obtain sufficient tumor tissue for molecular analysis, there is interest in identifying less invasive methods to guide immunotherapy, such as the use of circulating tumor DNA to detect TMB, known as blood TMB. Circulating tumor DNA-mediated measurement of TMB in peripheral blood appears to be a promising strategy for obtaining an effective and independent biomarker for estimating response to immunotherapy. However, many questions remain unsolved, such as appropriate panel size and which variants to include .+ tumor infiltrating lymphocytes (TILs) or CD8A mRNA transcription by IHC is associated with significantly prolonged PFS after treatment with PD-(L)1 inhibitor; this association was enhanced by combining PD-L1 with protein and mRNA levels, suggesting that the integration of these biomarkers may increase their predictive value [+ levels and clinical outcome was examined, and there was a significant association between sustained efficacy and OS [+ T-cell-infiltrating tumors with limited sensitivity to ICI, characterized by low T-cell proliferation (Ki-67 in T-cells) and cytolysis markers (granzyme B in T-cells). On the other hand, ICI treated patients whose tumor contained high CD3+ T-cell with high Ki-67 and granzyme B have favorable prognosis, indicating that not all inflamed (hot) tumors are the same. Recently, similar findings were reported in patients with advanced solid tumors using an 18-gene inflammatory mRNA signature. The higher the inflammation score, the greater the sensitivity to pembrolizumab, but some tumors with higher scores were noticeably more resistant to treatment [+ TIL was a factor for good prognosis in these tissues. However, in adenocarcinoma where the effect of smoking was not clear, CD8+ TIL did not correlate with prognosis, and FOXP3 positive T-cells correlated with prognosis. In particular, in adenocarcinomas of non-smokers, the high accumulation of CD8+ TIL was a factor in poor prognosis. These CD8+ T-cells were mainly CD8+ FOXP3+ T-cells and CD8+ GATA3+ T-cells, and the expression of CCR4 and CCL17 was also high in the tumor micro-environment. These results suggest that a negative immune environment is more likely to form in adenocarcinoma tissues of nonsmokers, which may be one of the reasons why ICI is less effective. Such analysis may also help to refine current strategies for individualized immunotherapy, including ICIs for NSCLC.Measurement of T-cell and other immune cell populations in tumor tissue is being actively developed as a predictor and endpoint for pharmacodynamic biomarkers and post-operative survival. In a variety of tumor types, the presence of significant baseline T-cell infiltration and interferon gamma (IFNγ)-related mRNA signatures (indicative of increased adaptive anti-tumor responses) has consistently been associated with increased sensitivity to ICIs and improved prognosis ,78,79,80ve value . In anoty and OS . In addireatment . There areatment ,84. In NAlthough a variety of other biomarkers have been reported, the precise biomarker for ICI has not been identified at this time. Standardization of the best assays and platforms to find reliable predictive biomarkers for immunotherapy is ongoing, and prospective studies are needed to demonstrate its value.Nivolumab showed an OS and PFS advantage over docetaxel in 2015 ,7. This In general, immunotherapy resistance is classified as primary resistance or acquired resistance . PrimaryOn the other hand, there are various immune and stromal cells and cytokines within tumor micro-environment, which can affect the response to immunotherapy. The changes of immunosuppressive cells, immunosuppressive cytokines, coinhibitory receptors, and costimulatory receptors in the tumor micro-environment can destroy the anti-tumor immune response, which are the important mechanisms mediating resistance to immunotherapy. Such acquired immune cell-dependent mechanisms have also been linked to ICI resistance, including insufficient T-cell activation , upregul+ T-cells [+ T-cells that will respond. This is one of the reasons adoptive immunotherapy using T-cells obtained from tumors was first used in lung cancer [Other immunotherapies for lung cancer have also failed ,111. Rea T-cells , and ine T-cells . There mg cancer . The faig cancer ,118. On g cancer . Finallyg cancer .In fact, it is important to obtain pre- and post-treatment tissue samples to determine what is happening in tumors that have become resistant to immunotherapy for lung cancer. There are a variety of approaches to studying the use of genetic testing to predict immunotherapy resistance. In NCT04300062, tumor tissues were collected at the time of progression during ICI therapy to ensure the study of molecular mechanisms involved in the progression of NSCLC and SCLC. By comparing the treatment outcomes of patients with specific molecular characteristics, NCT03512847 discovered a molecular profile that predicts resistance to immunotherapy and chemotherapy. Differences in molecular profiles before and after treatment may reveal mechanisms of resistance to treatment. A significantly higher likelihood of altered PD-L1 expression was observed in patients who received chemotherapy . In the Other immunotherapeutic approaches, including chimeric antigen receptor (CAR) and CD3-based bispecific agents, have been developed recently ,124. CARAn interesting aspect of adoptive immunotherapy for NSCLC is the use of glypican-3 (GPC3)-targeted T lymphocytes. Findings include, first, immunohistochemical assays showed that GPC3 was expressed in 66.3% of lung squamous cell carcinoma samples and 3.3% of lung adenocarcinoma samples, but not in normal lung tissue. Next, in two established lung squamous cell carcinoma xenograft models, CAR-GPC3 T-cells almost completely eliminated the proliferation of GPC3-positive cells, indicating that CAR-GPC3 T-cells are capable of detecting lung tumors and efficiently infiltrating cancerous tissues. GPC3 may be a promising target for the treatment of lung squamous cell carcinoma .As described above, there are various limitations in the current immunotherapy for lung cancer. It is necessary to combine the current immunotherapy with basic research  so that it can be an effective treatment for more patients with advanced lung cancer. For example, the cGAS-STING pathway, one of the innate immune pathways, has been reported to be associated with ICI sensitivity, both at in vitro model and in pre-clinical models. Kitajima et al. reported that STING expression is low in lung cancer with both KRAS mutation and LKB1 loss (KL tumor), and that the low STING expression level is caused by methylation of the promoter region of the STING gene . They shAn interesting, and potentially very attractive, new approach is to take a personalized approach to immunotherapy by harnessing the power of genetic sequencing. Although it is well known that cigarette smoking is associated with lung cancer carcinogenesis, there are many non-synonymous mutations in smoking-related lung cancers that have a molecular smoking signature , and can+ T-cells [Successful immunotherapy of lung cancer requires a better understanding of immune escape and immunosuppression in lung cancer, and learning how to monitor immunity, edit immunity, and reactivate immunity in cancer. By learning to understand cancer immunosurveillance, immunoediting, and how to reactivate cancer immunity, we can embark on a future full of possibilities to use immunotherapy as a reliable lung cancer treatment. The logical combination of nivolumab and ipilimumab in melanoma and NSCLC looks promising, although there are considerable side effects . Several T-cells ,142. In  T-cells . Prelimi T-cells . There a T-cells ,144,145. T-cells . The lon