From the beginning of public vaccinations until the relaxation of COVID-19 measures, many case reports, case series and case–control studies have been published indicating cutaneous side effects of COVID-19 vaccination. Post-vaccination pustular eruption was reported as well, with a challenging differential diagnosis between pustular psoriasis, AGEP  and neutrophil pustular eruptions. We report a case of 56-year-old woman presented with acute generalized pustular flare up culminated 5 days after the second dose of BNT162b2(Pfizer) vaccination. She was diagnosed with pustular psoriasis flare and due to the regulating role of IL-1 in pustular psoriasis and in the cytokine storm observed in cases of COVID-19 postvaccination inflammation; we decided to treat the patient with an IL-1 antagonist, subcutaneous anakinra (100 mg daily) along with acitretin. One week later, after anakinra withdrawal, she presented a pustular psoriasis flare and a 7-day anakinra re-administration led to a satisfactory improvement in the skin lesions. We also reviewed the medical literature and found 28 case reports with pustular eruption after the COVID-19 vaccination. We compared the patients reported, regarding sex, age, number of doses, post-vaccination period and vaccine brand, and compared those results with our patient. Finally, as indicated by our case and other cases with similarly treated pustular eruptions. targeted therapy to this cytokine imbalance such as anakinra (IL-1) antagonist can improve the clinical course of the patient. At present, the most efficient method for controlling and preventing epidemics, limiting the severity of the respective disease, reducing hospitalizations, and lowering the mortality rate is vaccination. COVID-19 vaccines including messenger RNA (mRNA) (Pfizer-BioNTech and Moderna) and viral vectors (Johnson & Johnson’s Janssen and AstraZeneca) were effective measures against COVID-19 pandemic  However,Pustular skin eruption was a type of post-COVID-19 vaccination exanthem that was referred to in the medical literature and often included diagnosis of pustular psoriasis (new onset or flare) , acute gThe exact immunopathogenesis link that connects the immune reaction mediated by the vaccine and the pathophysiology of the aforementioned pustular diseases remains obscure. Vaccination is proven to lead to a systemic immune activation resembling a cytokine storm with the production of various pro-inflammatory cytokines that participate in the pathogenesis of pustular dermatosis . In caseHerein, we present a case of a patient that developed pustular psoriasis flare after the second dose of mRNA COVID-19 vaccine who was successfully treated with IL-1 antagonist (anakinra) and we attempt to critically review the published case reports and case series of COVID-19 vaccine-associated pustular eruptions to aid proper and accurate diagnosis and efficacious treatment.2) came to the Emergency Department of the University General Hospital of Larissa, Central Greece with acute generalized pustular flare up that occurred 5 days after the second dose of BNT162b2(Pfizer) vaccination. The prior four months, she was under treatment with methotrexate (10–15 mg/week) for plaque psoriasis control plus conventional filicine supplements. Three days after the first dose of the vaccination, the patient reported the appearance of small pustules on the abdomen and thighs. She was initially treated with topical corticosteroid preparations but with moderate response. The fifth day after the second dose of her vaccination, the patient presented with painful erythematous skin patches with pinhead-sized sterile pustules, mainly located on the back, upper arms, abdomen area, thighs, soles and palms  occurring with a mean of 4-5 days after vaccination. Sex distribution was male dominant (n = 7) and age range was 18 to 72 years old. The most prevalent vaccine type regarding pustular psoriasis flare was Pfizer (n = 7). This flare occurred in most of the times after the first dose (n = 6). The new-onset pustular psoriasis eruptions were less (n = 3) than the flares. Neutrophilic generalized pustular dermatosis was also observed (n = 3) with more severe clinical presentations. Worth mentioning was the case of [We found 16 patients reporting a generalized pustular eruption after COVID-19 vaccination . Most of case of , which dFour of the cases ,16,17,18n = 3, rosacea-pustulosis n = 2, facial pustular neutrophilic eruption n = 2, eosinophilic pustular folliculitis n = 1 and acute localized pustulosis n = 1). Although the categorization of facial pustular neutrophilic eruption in the pustular dermatosis list is unclear, we considered that disease in the localized pustular dermatosis due to its face-specific distribution. Amongst the patients, sex was almost equally distributed  and the age range was 38 to 80 years old. Most of the eruptions occurred the fourth or fifth day after the vaccination, with two cases occurring from the same article referring a postvaccination period of one month [n = 5) and Moderna (n = 3) vaccines. A specific dose (first or second) did not seem to contribute more to the development of such dermatosis. The patients, apart from the pustules, presented with the classical characteristics of the respective disease, for example erythema in rosacea-like pustular eruptions, and were treated with the classical disease-specific therapy. Half of the cases (4 out of 8) were new-onset skin manifestations, while palmoplantar psoriasis was due to exacerbation of a psoriasis subtype. Interestingly, an acne vulgaris flare was not detected. Worth mentioning is that, despite the localized distribution of pustular eruption, there are diseases with different pathophysiology origins and therefore occlusive conclusions cannot be drawn. Also, Regarding localized pustular dermatosis after COVID-19 vaccination , we obsene month . The resp = 0.69), age (p = 0.78) and dose (p = 0.68) revealed no statistical importance, mainly due to a small amount of patients.Below are presented bar charts that compare sex, age, type of dose and vaccine brand in association with AGEP, pustular psoriasis and neutrophilic pustular dermatosis. Worth mentioning is the dominance of male sex in the disease cases, the prevalence of first-dose causality and Pfizer as the main type of vaccine causing pustular disease, especially in the case of pustular psoriasis . HoweverIn the beginning of the public vaccinations, the study of the cutaneous side effects of COVID-19 vaccination was limited, with only a few studies to have a large number of patients and focus on different dermatology conditions . As timeAGEP has a challenging differential diagnosis, which depends on both clinical and histopathology characteristics . Cases rIn addition to COVID-19 post-vaccination dermatoses, case reports regarding pustular diseases during the viral infection itself have been reported. Pustular psoriasis flare  as well Even though our patient was female, the conclusive results of our review showed a male dominance in pustular eruption cases. This sex distribution was also showed by Megna et al., who studied psoriasis flare ups after COVID-19 vaccinations . Since tAn increasing number of studies are providing additional information regarding the immunological response of COVID-19 vaccines and attempting to elucidate the development of post-vaccine skin manifestations, including pustular eruptions. It is hypothesized that inflammatory cytokines release such as IL-1, IL-6 and TNF-a seem to contribute to the formation of pustule or general to skin flare . The COVContrary to the more classic treatment options used in the case reports discussed, in order to control pustular psoriasis flares in our patient we used an IL-1 antagonist, anakinra. Anakinra is a regimen that large studies have indicated does not show great efficacy in pustular psoriasis subtype  and it iOur study presents some limitations. Firstly, there are categories presented in which the relevant studies and cases did not reach a statistically significant result, and secondly, only one medical database was used (Pubmed). Also, the concept of post-vaccination pustular eruption is new and therefore more case reports are anticipated to be published, giving new data that may not be included in the review.Pustular eruption followed by vaccination can be a challenging diagnosis including pustular psoriasis, AGEP and neutrophilic dermatosis. Similarities in vaccine brands, post-vaccination flare timing and patient-related characteristics cannot further direct the diagnosis, making the clinical and histopathological disease features the main indicators. The cytokine imbalance caused by vaccination can possibly trigger the eruption, but studies on cytokine measurements on dermatology patients should be performed to confirm this hypothesis. Targeted therapy to this cytokine imbalance such as anakinra (IL-1) antagonist can improve the clinical course of the patient.


EGFR alterations, the clinical efficacy of EGFR-TKIs in these discordant cases remains little known. We retrospectively reviewed consecutive patients with non-small-cell lung cancer whose FFPE samples were simultaneously submitted for the ODxTT, and a PNA-LNA PCR clamp test. We evaluated the clinical efficacy of EGFR-TKIs in patients with discordant results between the two tests, focusing on the common EGFR mutations. Among 444 successful results, 10 patients had discordant results for common EGFR mutations (9 Ex 19 deletion and 1 Ex 21 L858R mutation), and all of these were detected only by the PNA-LNA PCR clamp test. Among six discordant cases treated with EGFR-TKI, the mutations detected in 3 patients were not included in the list of detectable variants that are reportable by the ODxTT, while the mutations detected in the other 3 patients were included in the list. For all three discordant cases harboring the mutations not reportable by the ODxTT, good clinical responses were demonstrated. However, among the other three discordant cases harboring the mutations reportable by the ODxTT, only one patient had a clinical response with short duration. Among the discordant cases for common EGFR mutations between the ODxTT and the conventional single gene test, there are a certain number of suitable patients responsive to EGFR-TKIs, especially when the cause of the discordant results comes from the difference in the range of detectable variants that are reportable between the tests.Although we have experienced some cases with discordant results between the Oncomine Dx target test (ODxTT) and conventional single gene tests for detecting  Clinical guidelines therefore recommend that patients with advanced NSCLC harboring driver oncogene alterations receive targeted therapies for each alteration as the first-line treatment8. Among the driver oncogene alterations, epidermal growth factor receptor (EGFR) alterations are most commonly detected, occurring in 45% of patients with advanced non-squamous NSCLC in Japan9.Various molecular-targeted drugs for patients with non-small-cell lung cancer (NSCLC) harboring driver oncogene alterations have improved patient prognoses better than conventional cytotoxic chemotherapyEGFR mutations, such as the cobas EGFR assay, the therascreen EGFR assay as an in vitro diagnostic (IVD) test, and the peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp assay as a laboratory-developed test (LDT), have been widely used as single companion diagnostic tests for EGFR-tyrosine kinase inhibitors (TKIs)12. However, as more target genes have been identified and targeted therapies have been approved in clinical settings, comprehensive biomarker testing has become necessary to make appropriate treatment decisions.Conventional single-gene tests for 13. Since February 2019, this test has also been approved in Japan as a companion diagnostic for targeted therapies on 4 driver alterations: EGFR mutations, ALK fusion genes, ROS1 fusion genes, and BRAF mutation (p.V600E). In addition, RET fusion genes have been added to the ODxTT as a companion diagnostic in Japan since September 2021.Next-generation sequencing (NGS) can detect multiple gene variants simultaneously, enabling comprehensive genetic testing. The Oncomine Dx target test (ODxTT)  is one of the NGS panels, and was approved by the US Food and Drug Administration in June 201714, the clinical efficacy of EGFR-TKIs in these discordant cases remains little known. Therefore, in this study, we retrospectively evaluated the clinical efficacy of EGFR-TKIs in patients with discordant results between the two tests, focusing on the common EGFR mutations (Ex 19 deletion and Ex 21 L858R), taking into account the identified variants of the mutations.Although we have previously reported some cases with discordant results between the ODxTT and the PNA-LNA PCR clamp assay for the detection of EGFR mutationsEGFR mutations of each test for adenocarcinoma, are shown in Fig. A total of 470 samples were identified for comparison analysis. The sample characteristics for the analysis are shown in Table Among the 444 samples successfully analyzed by both tests, 10 discordant results for common EGFR mutations were reported, whose clinical characteristics and courses are shown in Table Six of the ten discordant cases of common EGFR mutations were treated with EGFR-TKIs , all of whom received the treatments as first line therapy. Among the three treated patients harboring the common EGFR mutations not reportable by ODxTT, all of them had good clinical responses. However, among the other three treated patients harboring the common EGFR mutations reportable by ODxTT, only one patient had clinical response with short duration. PFS and TTF of the patients harboring the common EGFR mutations not reportable by ODxTT were longer than those of the patients harboring the common EGFR mutations reportable by ODxTT Fig. . Of the 16, the clinical utility of targeted therapies for these discordant cases remains little known. To our knowledge, this is the first report to assess the clinical efficacy of EGFR-TKIs in patients with discordant results for common EGFR mutations between the ODxTT and a single gene test, focused on the range of detectable variants in each test.In recent years, multi-gene tests able to detect multiple driver alterations simultaneously, such as NGS and PCR panel tests, have become available in clinical practice. Although we have experienced some cases with discordant results between the multi-gene tests and conventional single-gene tests in such situationsEGFR Ex 19 deletion, and 8% AF for Ex 21 L858R17. A clinical bridging study to establish the assurance of the ODxTT compared with the therascreen EGFR assay, whose LODs were 1–2% for EGFR common mutations, was performed by Thermo Fisher Scientific and showed good concordance17. Meanwhile, the LODs for the PNA-LNA PCR Clamp test are estimated to be 0.1–1%11, therefore it is expected that the PNA-LNA PCR clamp test can detect lower AFs for EGFR mutations. If the discordant results come from the difference in LOD between the tests, the tumors harboring the mutation would not be predominant in the whole tumor when the assumed tumor content was not overestimated, therefore poor EGFR-TKI efficacy is expected in these cases. If the discordant results come from the difference in the range of detectable variants of each test the tumors harboring the mutation may be predominant in the whole tumor, therefore good EGFR-TKI efficacy is expected in these cases. Among the three patients in this study treated with EGFR-TKIs harboring common EGFR mutations reportable by ODxTT, this indicating the discordant results were due to the difference of LOD between the both tests, only one patient had a clinical response with short duration. Meanwhile, among the three patients treated with EGFR-TKI, harboring Ex 19 deletion variants not reportable by ODxTT, this indicating the discordant results were due to the difference in the range of detectable variants between the tests, all of them had good clinical responses with long duration.In this study, all common EGFR mutations in the discordant results were only detected with the PNA-LNA PCR clamp test, and we additionally ordered the laboratories to perform the direct sequence method using reaction products after the PNA-LNA PCR clamp assay to identify the COSMIC ID of the specific variants. As for the cause of the discordant results, two main reasons should be considered. As shown in our previous report, one is the difference in limit of detections (LODs) between the ODxTT and the PNA-LNA PCR Clamp test, and the other is the difference in the range of detectable variants for each test. The estimated LODs of the ODxTT were reported to be 6% AF for 19. Although the efficacy of EGFR-TKIs for uncommon (non-ΔE746_A750 deletion) Ex 19 deletion variants is controversial, some previous reports have demonstrated good efficacy of EGFR-TKIs among patients harboring uncommon Ex19 deletion variants21. Our results suggested that EGFR-TKIs would be a suitable treatment option for patients harboring uncommon Ex19 deletion variants, and missing suitable patients responsive to EGFR-TKIs due to the difference in range of detectable variants for each test would be an important issue to be avoided. Therefore, the ability of a test to detect and report an extensive range of mutation variants would be a desired factor in optimal genetic testing.EGFR Ex 19 deletions consist of distinct molecular variants and represent a heterogeneous disease entity, of which the most common Ex 19 deletion variant is ΔE746_A750 deletion, detected in 65–72% of patients with EGFR Ex 19 deletion variants22. Therefore, it is difficult to precisely evaluate the relation between clinical benefit and tumor content in this study, although the tumor content plays a large role in the interpretation of the AF. Third, this study could not adequately evaluate other predictors potentially associated with EGFR-TKI efficacy, such as concurrent genetic alterations including TP53 mutation and PTEN alteration23, because the ODxTT could not evaluate these genes. Further studies, including evaluation for such predictors with larger genetic panel tests, would be needed. Finally, the range of detectable and reportable mutation variants was different for each single-gene and multi-gene test; therefore, the results of this study are not applicable to other single-gene and multi-gene tests. In conclusion, among the discordant cases between the ODxTT and conventional single-gene tests, a certain number of suitable patients are responsive to EGFR-TKIs, especially when the discordant results come from the difference in the range of detectable or reportable variants in each test.There were several limitations to this study. First, this study was a relatively small retrospective study, which resulted in an analysis with a small number of cases. A larger sample size would be needed to evaluate the frequency of discordant cases, and the clinical meaning of detecting the discordant cases considering the clinical benefit of targeted therapies. Second, tumor content was evaluated subjectively by skilled cytopathologists in our institution, but not objectively by artificial intelligence or other means. Previous reports suggest that pathologists often overestimate the contents of tumor cells, and the use of AI-based analysis increases the accuracyThis retrospective study was conducted at Matsusaka Municipal Hospital, Japan. We reviewed the electronic data from consecutive NSCLC patients whose formalin-fixed and paraffin-embedded (FFPE) samples were simultaneously submitted for an ODxTT, and a PNA-LNA PCR clamp test, using the same specimen, from August 2019 to March 2022. Samples collected in other hospitals, and archived samples were excluded. Clinical data assessments included; patient characteristics, sampling methods, staging, histology, pathological findings, the results of genetic tests, and the clinical course with EGFR-TKI treatment. This study was performed in accordance with the Declaration of Helsinki. This study was approved by the institutional review board of Matsusaka Municipal Hospital (IRB number J-76-200410-5-2). Informed consent was obtained by opt-out method.15. The amount of tumor cells, and the tumor content of the sample stained with hematoxylin and eosin, were evaluated by skilled cytopathologists. In some samples obtained since 2020, macro-dissection was performed as needed in our institution. If the tumor content was < 20% after marking and macro-dissection, or the amount of tumor cells was insufficient, the sample was not submitted for the ODxTT. For the ODxTT, 10 to 20 slide-mounted 5 to 10-µm sections of small biopsy samples, and 5 to 10 slide-mounted 5 to 10-µm sections of surgical resection samples, depending on each sample volume, were submitted to LSI Medience Laboratories . For the PNA-LNA PCR clamp test, 5 slides of 5-µm sections from small biopsy samples and surgical resection samples were submitted to the laboratories. LSI Medience Laboratories performed the ODxTT based on Thermo Fisher’s Ion AmpliSeq technology, and the PNA-LNA PCR Clamp tests were performed using the PNA-LNA PCR clamp assay13. When a discordant result whose EGFR mutation was only detected by PNA-LNA PCR clamp assay was returned, we additionally ordered the laboratories to perform the direct sequence method using reaction products after the PNA-LNA PCR clamp assay to identify the specific variants of EGFR mutations.The FFPE sample processing methods of our institution were shown in detail in our previous reportsEGFR mutations detectable by the ODxTT and PNA-LNA PCR Clamp test were mentioned in our previous report14. In addition, the range of detectable EGFR mutation variants for the ODxTT is publicly available, and shown in Supplemental Table The specific EGFR mutations for the ODxTT compared with those of the PNA-LNA PCR clamp test in the same manner as shown in our previous report14. In this study, focused on the NSCLC patients with discordant common EGFR mutation results between the ODxTT and the PNA-LNA PCR clamp test, we evaluated their clinical response, progression-free survival (PFS), and the time to treatment failure (TTF) of EGFR-TKIs.We evaluated the success rate and the detection rate of EGFR mutations reported for each time period were completely available; exon 18 (p.G719A/C/S), exon 19 (deletion), exon 20 , and exon 21 (p.L858R and p.L861Q). These being the mutations considered required for detection due to clinical implications by the Japanese Lung Cancer Society. Meanwhile, the analysis results were regarded as unsuccessful if the sample did not pass the nucleic acid concentration threshold, or if one or more of the EGFR mutation results mentioned above were invalid due to a failure to meet the DNA sample quality control (QC) metrics, or no call.The analysis results were regarded as successful if all results for the following Statistical analyses were performed using Pearson’s Chi-squared test for comparison of analysis success rates and detection rates, and 95% Clopper–Pearson confidence interval (CI) were calculated. Statistical significance was indicated with a P-value less than 0.05. Statistical analyses were performed using SPSS software, version 26.0 .Supplementary Table 1.


Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit ( Taeniopygia guttata) already 25 years ago (Riparia diluta) and in sand martins (Riparia riparia), both females and males can form bivalent and univalent GRCs  was discovered in the zebra finch  suggested that many GRC genes are likely truncated pseudogenes —has been widely studied, including its adaptive history using genome scans , near Seewiesen, Bavaria, Germany, and flash-froze the testis, ovary, and blood. Samples were kept directly (testis and ovary) or in ethylenediaminetetraacetic acid tubes (blood) at −80 °C. High-molecular weight DNA  and total RNA (testis and ovary) were extracted at the Uppsala Genome Center, Uppsala, Sweden, using the Monarch HMW DNA extraction kit (DNA) and the TRIzol reagent and Phasemaker tube extraction system (RNA). At the same center, the samples were sequenced using long-read PacBio HiFi technology (Sequel II) after library preparation with the SMRTbell Express Template Prep Kit (according to the Iso-Seq protocol for the RNA sample). The testis DNA sample was run on three SMRT cells , the blood DNA sample on one SMRT cell , and the testis and ovary RNA samples on two SMRT cells each . The Iso-Seq long-read data were processed using the SMRT Link pipeline and used to characterize and map the full-length transcriptome of the adult testis and ovary tissue.We estimated expression of all the characterized full-length transcripts using the previously published RNA-seq data from First, we assembled a new somatic reference genome from the long-read data of the male blue tit blood sample using hifiasm . The assA, supplementary fig. S1B, Second, as described below, we extracted and assembled reads with germline-specific alleles from the testis sequencing data to build a GRC of contigs that were sufficiently different from the A-chr contigs. Our method was designed to identify and compare sufficiently different paralogous genes of the GRC and the A-chrs for downstream differential expression analyses. Thus, we mapped reads of the testis (germline) sample and the blood (soma) sample of the same male individual on the new soma reference genome using minimap2 with permissive parameters  to allow testis reads of potential GRC origin to map to homologous A-chr regions . Mappingsupplementary fig. S1A and B, To validate the assembled GRC contigs, we mapped soma and germline reads on the combined genome assembly (A-chr plus GRC) using minimap2 in a stringent setting. Median soma read depth in A-chr and GRC windows of 25-kb size was 30× and 0.4×, respectively, while the median germline read depth on the A-chr and GRC was 88× and 24×, respectively. The higher A-chr coverage in the testis compared with the blood (88× vs. 30×) reflects the higher sequencing effort in the testis sample (median ratio of coverage = 2.87). The difference between the GRC and A-chr coverage within the testis sample (24× vs. 88×) is expected, given that the GRC occurs only as a haploid and only in the spermatocytes of the sampled testis cells. The ratio of the GRC to A-chr coverage (0.27) is consistent with past studies sequencing testes from other songbirds . We furtsupplementary fig. S2We mapped the 400,403 and the 508,427 Iso-Seq full-length transcript sequences from the testis and ovary sample, respectively, as well as the 42,986 National Center for Biotechnology Information (NCBI)-annotated transcript model sequences of the previously published blue tit genome on our assemblies using minimap2. We included the NCBI–annotated transcripts based on RNA-seq data from different tissues including the brain, gonads, and a diverse set of organs, to expand the transcriptome beyond the testis and ovary. We mapped the transcriptome separately on the A-chr and GRC assemblies to characterize paralogous genes with high likelihood both on the GRC and on the A-chr. Transcript sequences that redundantly mapped to the same genomic location were collapsed using tama collapse , resultiLuscinia megarhynchos and Luscinia luscinia) from a previous study  sets and PANTHER Protein Class annotations . This lius study  to evaluThe sequence completeness of protein-coding genes was evaluated by inspecting the output of the BlastX mapping against the UniProtKB database (see above). We compared the 83 gene symbols of the paralogous genes between GRC and A-chrs by subtracting the respective median values across all isoforms and multiple gene copies within the GRC and A-chrs, respectively.We quantified the expression of all isoform models in the A-chr and GRC (see above) based on the RNA-seq data of the five testes and the five ovary samples . To do tThe GRC of the two sampled blue tits has the size of a microchromosome, as shown by cytogenetic techniques . In lineWe characterized 564 genes in the GRC assembly based on full-length transcriptome data, of which 244 showed consistent mapping with one of the UniProtKB proteins and are thus potentially protein coding. The residual set consists of 320 genes that are presumably either noncoding or else unable to match one of the 205,788 known protein sequences, because they are too short or divergent. A gene symbol could be assigned to 164 of the protein-coding genes resulting in 103 different symbols. The percentage of sequence occupied by protein-coding and noncoding genes appears to be higher on our GRC assembly than on the A-chr assembly  online.r = 0.35; n = 83; P = 0.001; ERVs) and other known avian multicopy genes  appear in multiple copies both on the GRC and on the A-chrs, whereas other genes like EPS15L1 and BMP15 have multiple copies only on the GRC. Most paralogous genes, as represented by GRC gene symbols, have a similar or slightly lower median number of isoforms in the GRC than in the A-chrs with PIM1, PAK1, and MROH1 as the strongest exceptions to this rule  and SIX6OS1 (blue tit only).To test whether the detected enrichment of terms on the GRC is a general phenomenon, we additionally analyzed the gene content of the GRC in two nightingale species . The 84 BMP15, EPS15L1, B9D2, and PFKM show >10-fold higher TPM values in GRC than in A-chr paralogs in at least one sex; BMP15, which shows a slight female-biased expression on the A-chr but a much stronger female-biased GRC expression. Other genes with strong female-biased expression are H1-8 and PIM3 -induced progesterone production from granulosa cells of large maturing preovulatory follicles . Such genes might be responsible for the maintenance of the GRC over millions of years. The synaptonemal genes may have been important in shaping the GRC inheritance patterns, either by maintaining the predominant behavior of maternal inheritance or by allowing or facilitating episodes of alternative inheritance patterns.In summary, we suggest that the genes shared among the characterized GRCs of songbirds, including msad096_Supplementary_DataClick here for additional data file.


Heart failure remains a leading cause of hospitalization and death, and presents a significant challenge for healthcare providers despite the advancements in its management. This umbrella review aimed to pool the results of meta-analyses on the use of sodium-glucose cotransporter-2 (SGLT-2) inhibitors in the treatment of heart failure patients. A literature search was done on five databases: PubMed, Cochrane Library, Scopus, Global Index Medicus, and Science Direct for articles with full texts available online. Meta-analyses of five or more randomized controlled trials (RCTs) were included; the assessment of multiple systematic reviews (AMSTAR) was used to assess the quality of included studies. A systematic search identified 10 relevant meta-analyses of RCTs, with primary analyses including outcome data from 171,556 heart failure patients. A pooled review showed that SGLT-2 inhibitors significantly reduced the risk of heart failure hospitalization, cardiovascular death, mortality, serious adverse events, and improved quality of life. SGLT-2 inhibitors are likely safe and effective in managing patients with heart failure especially considering the acute outcomes. Heart failure is a chronic ailment that is characterized by the heart's inability to pump blood efficiently, and some of its symptoms are fatigue, shortness of breath, and fluid retention in the body . With itIn addition to its advantages for treating type 2 diabetes, sodium-glucose cotransporter 2 (SGLT-2) inhibitors have recently become a promising therapeutic option for treating patients with heart failure . SGLT-2 The objective of this umbrella review is to summarize the results of meta-analyses on the use of SGLT-2 inhibitors in the treatment of heart failure patients and to offer a comprehensive evaluation of the efficacy and safety of these drugs in this population. This review provides a thorough summary of the available research and is helpful to policymakers and healthcare professionals in making decisions about the use of SGLT-2 inhibitors in the treatment of heart failure.MethodsThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, according to Liberati et al., were followed while conducting this study .Search methodsThe literature was searched for topic-related articles using five databases: PubMed, Cochrane Library, Scopus, Global Index Medicus, and Science Direct looking for the search terms like sodium glucose co-transporter-2, SGLT-2, heart failure, and cardiac failure. Additional keywords and the full search strategies are outlined in Table Eligibility criteriaThe criteria for inclusion of a study were as follows: systematic review and meta-analysis (SR-MA) studies, SR-MAs with atleast five or more randomized controlled trials (RCTs), and papers in the English language. Given the amount of literature available on the topic, we determined that a meta-analysis with at least five studies would help avoid poor-quality SR-MA studies. The criteria for exclusion of the study were as follows: systematic reviews that did not pool data or carry out meta-analysis; narrative reviews; studies of the efficacy or safety of SGLT-2 inhibitors in patients that did not have heart failure; studies that did not compare SGLT-2 inhibitors with placebo and only evaluated individual factors in heart failure patients such as age, diabetes status, gender, or baseline therapy.With a larger number of primary studies, the statistical power of the analysis increases. This means that the umbrella review can provide more robust and reliable conclusions by pooling data from a larger sample size. The inclusion of SR-MAs with five or more primary studies in an umbrella review will lead to narrower confidence intervals and hence more precision. This increased precision allows for more accurate estimation of the outcomes of patients taking SGLT-2 inhibitors. Study selectionTwo authors independently assessed the titles and abstracts of papers to determine if they met the inclusion and exclusion criteria. After entering each reference in Endnote software , duplicates were eliminated. Every meta-analysis that satisfied the requirements for inclusion had its reference lists manually searched to make sure no pertinent study was overlooked. The entire text was examined in cases when abstracts did not provide enough information to determine if inclusion or exclusion criteria were met. Every meta-analysis that satisfied either set of criteria was carefully evaluated before the final inclusion decision was made, and any differences between the two independent reviewers were settled in a consensus meeting with a third reviewer.Data extractionThe following data was extracted from each of the included study: the primary author, the journal, the year of publication, the search range in years of studies that were included in the meta-analysis, and the number of primary studies that were included in the meta-analysis. Date range of the primary studies that were part of the meta-analyses were listed along with the following: the sample size of the study, the population it was drawn from, the meta-findings, analysis such as the mean difference and standardized mean difference for the risk ratio, confidence intervals (CI), the p-value, and the classification of heart failure based on ejection fraction. Assessment of methodological qualityThe internal validity of the meta-analyses included in this research was evaluated using the assessment of multiple systematic reviews (AMSTAR) tool . In a syDegree of primary study overlapThe corrected covered area (CCA) created by Pieper et al. can be used to quantify overlap in primary research included in reviews . By "divResultsStudy SelectionThe electronic database search provided an initial 841 studies. A manual search of relevant journals and reference lists provided one study. Sixty-one duplicates were removed from the 842, leaving 781 articles from which a title and abstract screening removed 731 as not relevant. The full text of all remaining 50 articles was downloaded to be assessed in detail; 40 of these were excluded in the detailed assessment and 10 meta-analyses were included in the meta-review. Figure Characteristics of Included StudiesTable The reviews included a total of 27 primary studies, and the meta-analyses used a minimum of five and a maximum of ten studies Table . No consMethodological Quality AssessmentAMSTAR 2 rated eight studies as being of moderate quality, one study as being of high quality, and one study as low in quality. While limitations were mentioned, none of the included meta-analyses addressed the possible impact of risks of bias (in their study) in a quantifiable manner; half failed to carry out an adequate investigation of publication bias, while only one of the ten provided a list of excluded studies Table .DiscussionIn order to demonstrate the efficacy and safety of SGLT-2 inhibitors for patients with heart failure, this study was conducted to provide a systematic aggregation of evidence from several meta-analyses into a single, current database that is easily accessible. Ten meta-analyses were incorporated into the review, and the combined findings of this study showed that SGLT-2 inhibitors are linked to significantly lower rates of cardiovascular death or heart failure hospitalization in patients with heart failure, cardiovascular death, all-cause mortality, and heart failure hospitalization outcomes. However, two meta-analyses revealed no significant difference in cardiovascular death and all-cause mortality outcomes between SGLT-2 inhibitors and placebo ,7.Most studies discovered that the use of SGLT-2 inhibitors may result in better outcomes for individuals with heart failure, including decreased rates of hospitalization and death from cardiovascular causes. And since SGLT-2 inhibitors are also successful at controlling glucose levels in people with type 2 diabetes, this evidence will be especially helpful for people with heart failure who also have uncontrolled diabetes. Additionally,looking at most of the studies, SGLT-2 inhibitors were also linked to less serious adverse events and a higher improvement in Kansas City Cardiomyopathy Questionnaire (KCCQ) scores. In comparison to other heart failure medicines, SGLT-2 inhibitors may be associated with enhanced safety due to the decreased frequency of major side events and improvement in KCCQ score. This may result in better outcomes and a higher quality of life for heart failure patients as well as lower hospitalization and other medical intervention expenses -9,14-19.Results also highlight the necessity of compiling the meta-analyses into a single, comprehensive review to reflect the validity of the conclusions reached by each individual meta-analysis. That was the study's objective, and it is what the study ultimately attained. Because reducing cardiovascular death, heart failure hospitalization, and all-cause mortality leads to better performance and lower treatment costs; this work will have significant implications for health organizations as well as individuals with heart failure and medical professionals. This study's findings also suggest that SGLT-2 inhibitors may reduce serious adverse effects in addition to improving the quality of life. Either way, the net result is probably going to be less strain on the healthcare system. One major concern was that no study evaluated participants with advanced renal failure with GFR<20 mL/min/1.73 m2. Hence, it remains questionable whether the drug should be used in this subset of patients. The American College of Cardiology (ACC) expert consensus also has highlighted a similar problem where renal compromised patients may have attenuated effects to the use of this drug .The umbrella review has some limitations; multiple meta-analyses have overlapping primary studies between them (as illustrated in the citation matrix above), so the studies that appear in more reviews (the ones that overlap in multiple studies) could have a stronger impact on the findings of this review. More meta-analyses have been done on earlier published studies, which means they are probably overrepresented in the literature, leading to publication bias.Prior to this research, there had not been an umbrella review on the use of SGLT-2 inhibitors in the treatment of patients with heart failure. Overall, our review of the literature shows that SGLT-2 inhibitors can reduce the risk of cardiovascular death, all-cause mortality, and heart failure hospitalization when used in the treatment of heart failure. Most meta-analysis studies support the use of SGLT-2 inhibitors in the treatment of heart failure as SGLT-2 inhibitors are also linked with reduced serious adverse effects; however, its use in patients with severely impaired renal function remains an enigma.


The active pursuit of network medicine for drug repurposing, particularly for combating Covid-19, has stimulated interest in the concept of structural control capability in cellular networks. We sought to extend this theory, focusing on the defense rather than control of the cell against viral infections. Accordingly, we extended structural controllability to total structural controllability and introduced the concept of control hubs. Perturbing any control hub may render the cell uncontrollable by exogenous stimuli like viral infections, so control hubs are ideal drug targets.We developed an efficient algorithm to identify all control hubs, applying it to the largest homogeneous network of human protein interactions, including interactions between human and SARS-CoV-2 proteins. Our method recognized 65 druggable control hubs with enriched antiviral functions. Utilizing these hubs, we categorized potential drugs into four groups: antiviral and anti-inflammatory agents, drugs acting on the central nervous system, dietary supplements, and compounds enhancing immunity. An exemplification of our approach’s effectiveness, Fostamatinib, a drug initially developed for chronic immune thrombocytopenia, is now in clinical trials for treating Covid-19. Preclinical trial data demonstrated that Fostamatinib could reduce mortality rates, ICU stay length, and disease severity in Covid-19 patients.Our findings confirm the efficacy of our novel strategy that leverages control hubs as drug targets. This approach provides insights into the molecular mechanisms of potential therapeutics for Covid-19, making it a valuable tool for interpretable drug discovery. SARS-CoV-2 and its variants have infected more than 767 million people and claimed more than 6.9 million lives worldwide, as reported to WHO . The numbers are climbing despite several vaccines have been administrated in many countries. The viruses can penetrate the vaccines and spread rapidly in densely populated areas. Therefore, it is urgent to develop effective drugs for treating SARS-CoV-2 infection.The devastating Covid-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, and developing new drugs for Covid-19 is challenging5. One approach to shorten the period of finding effective medicine is to reposition or repurpose the drugs initially developed for other diseases, a major focus of drug discovery for Covid-199. However, the space for drug repurposing is enormous10. The most popular computational approaches for drug repurposing take the perspective of systems biology or network medicine16. Among these are eminent methods based on the well-established network structural controllability16. Following the theory of structural controllability18, the cell is regarded as a network of genes/proteins that can be controlled by exogenous stimuli  on a set of driver nodes  so that the cell can be driven from any state to the designated state in finite time. Structural controllability has been directly adopted to repurpose drugs for treating Covid-1916. Driver nodes targeted by existing drugs can give rise to putative reusable drugs, and the results were validated using bioinformatic methods and data in the literature16. Structural controllability has been applied to protein-protein interaction networks19, gene regulatory networks20, and metabolic networks21. The concept of driver nodes matches well with that of cancer driver genes22 because frequent mutations (which are viewed as stimuli) in such genes may induce tumorigenesis, so the concepts of structural controllability and driver nodes have been applied to finding cancer driver genes as therapeutic targets for precision cancer treatment23. Note that explaining explainability is a prominent feature of drug repurposing approaches as it provides insight into the inner workings of the drugs identified and boosts confidence in adopting the drugs against the target disease12.Drug discovery is notoriously costly and time-consumingWhile theoretically sound, straightforward application of structural controllability to drug repurposing is impractical. The key to the structural controllability of a network is a control scheme consisting of a set of control paths and their starting nodes (driver nodes or genes) that can be used to steer the network from any arbitrary state to the designated state in a finite time. Under this theory, a drug is used as an exogenous force to change the states of the cell, hopefully, from an infectious or cancerous state to a normal state. However, the normal states of a cell are typically unknown, so it remains unclear what external stimuli or drugs should be used. Moreover, the control scheme is not unique, and a control scheme typically has too many driver nodes to be practically manipulated at once to control the cell.control hubs, which are nodes residing on a control path of every network control scheme. The control hubs are the most vulnerable spots to the structural controllability of the network; a perturbation to any control hub may render the network uncontrollable by any control scheme. Therefore, control hubs are ideal drug targets for protecting the cell from exogenous influences. Moreover, exploiting control hubs as drug targets is a more practical approach for drug repurposing because control hubs are typically an order less than driver nodes, as to be shown shortly. Without computing all control schemes, which is a #P-hard problem24 , we developed a polynomial-time (meaning efficient) algorithm for finding all control hubs for a network. We applied our novel control hub-based drug repurposing approach to the largest homogenous human protein-protein interaction (PPI) network25 (Table S1), along with the data of PPIs between SARS-CoV-2 and human27 (Table S2) and the data of drug targets28 (Table S3), to discover control hubs that are targets of some existing drugs. Such druggable control hubs can not only be adopted for treating SARS-CoV-2 infections but also provide insights to explain the functions and mechanisms of the medicines in combatting the infection.We pushed the envelope of the theory of structural controllability. Instead of attempting to control the cell, we aimed at protecting the cell from viral infections. Specifically, we expanded our perspective from structural controllability by a single network control scheme to a global view of total controllability over all control schemes for the network. We then introduced the concept of We first outline the rationale of our novel control-hub-based method and present its primary steps. We then apply it to an integrated network constructed using human and SARS-CoV-2 PPI data and the data of drugs and drug targets. We compare our new method with nine existing gene selection methods, including the structural-controllability-based driver-node method, to show its performance in finding drug targets for Covid-19. We then examine the 65 drug targets and the corresponding drugs identified by our new methods, using the data and results in the literature for validation.18 is a control scheme for a network. It consists of control paths such that every node in the network can be reached or controlled by the head node of the control path to which the node belongs  to enter the host cell, triggering a series of adverse signaling cascades25 (Table S1) contains 4,529 driver nodes, which are 49.8% of the 9,092 nodes in the network. Determining the best or an effective control scheme is a daunting task.Moreover, it is impractical to directly adopt structural controllability for controlling the cell or repurposing drugs. The control scheme is not unique . An expocontrollability by considering all control schemes and introducing a new concept of control hubs. A control hub is a middle node in one of the control paths of every control scheme .Since the concept of control hubs is built atop all control schemes, a technical obstacle is the potentially exponential number of control schemes for a network. Finding all control schemes using the current best method, i.e., maximum matchingControl hubs can act as surrogates to reusable drugs, i.e., we focus on those existing drugs that can target control hubs. While in theory, any drug-targeted control hubs can be used, the ones closer to exogenous stimuli  are preferred over the distant ones since blocking the former may prevent the spread of external influences sooner and more effectively.25 (Table S1) and the data of PPIs between SARS-CoV-2 and human27 (Table S2) and the data of drug targets28 (Table S3). The human PPI subnetwork and the virus PPI subnetwork are linked through the PPI between human and virus proteins, and the human PPI subnetwork and drug subnetwork are connected by the drug target information. The resulting network contains 9,092 nodes (proteins) from humans, 22 nodes from SARS-CoV-2, and 2,980 nodes of drugs. The overall network is relatively tight, with a total of 81,953 links.We capitalized on total controllability and control hubs and developed a drug-purposing method consisting of four major steps  were drug targets  randomly selected proteins in the community should follow an empirical normal distribution . The driver-node method was slightly worse than the baseline model since the mean of the former was smaller than the mean of the latter  of SARS-CoV-242  directly interact with nonstructural proteins of SARS-CoV-227 , S4A. RI-CoV-242 , 3B. nspRS-CoV-2 , 3B, whi45. SARS-CoV-2 infection promotes the expression of RIPK1 in the lung of Covid-19 patients, and small-molecule inhibitors of RIPK1 can reduce the viral load of SARS-CoV-2 and proinflammatory cytokines in human lung organoids, indicating that the virus hijacks RIPK1-mediated immune response for its replication and propagation46. RIPK1 is targeted by Fostamatinib  showed that Fostamatinib could lower mortality, shorten the length of ICU stay, and reduce the disease severity of critically ill patients49.RIPK1 encodes serine/threonine-protein kinase 1, plays a role in necroptosis, apoptosis, and inflammatory response, and mediates cell death and inflammationamatinib , a drug 53. Indeed, coronavirus infection dysregulates the NAD metabolome, as indicated in a preclinical study54. Moreover, early phases 2 and 3 clinical trials showed that medication of NADH in a mixture of two metabolic activators could significantly shorten the time to complete recovery of SARS-CoV-2 infection55.CYB5R3 encodes NADH-cytochrome B5 reductase 3, a flavoprotein with oxidation functions. It is targeted by three drugs , S4A, tw56, as women are less susceptible to Covid-1958 and mice with weaker estrogen receptor signaling due to respiratory coronavirus infection exhibit increased morbidity and mortality59.COMT encodes catechol-O-methyltransferase that can degrade estrogens, catecholamines, and neurotransmitters such as dopamine, epinephrine, and norepinephrine. It is targeted by 14 FDA-approved drugs, including Conjugated estrogens , S4A. CoBeyond the three druggable control hubs that directly interact with viral proteins, 19 druggable control hubs in the 2-step community engage more than one viral protein via another protein, and four of them  are targeted by more than one drug  proliferation, cellular response to  stress, regulation of apoptotic signaling, and response to nutrient levels . All thehttps://clinicaltrials.gov/ct2/home). It is desirable to use drugs with multiple targets to gain treatment efficacy; the potency of a drug can be estimated by the number of control hubs it targets. Remarkably, 15 drugs target more than one control hub, and seven target more than two druggable control hubs , which entrap and eliminate pathogens during viral and bacterial infections and may cause adverse injury to surrounding tissues by themselves or by increasing proinflammatory responses60 , which encodes a soluble protein that is a form of vitamin E , 1) antiviral and anti-inflammatory agents that are subscribed for virus infection and cancer treatment, 2) dietary supplements including NADH and Calcium that boost human immunity, 3) hormones, including conjugated estrogens, and 4) drugs acting on central nerve systems. Combined, the medicines in the first three categories help boost immunity to overcome viral infections’ adverse stress and influence.16. Most of these network-biology methods hinge upon the idea that important proteins can be surrogates for identifying medicines. However, these methods operate under different notions of what constitutes important proteins in biological networks. For example, proteins with high degrees of connectivity may be considered essential since they supposedly affect many neighboring proteins.Network medicine for drug repurposing has gained popularity and momentum since the Covid-19 pandemic18 has been adopted as an approach to network medicine. Using driver nodes as drug targets is particularly appealing for Covid-19 drug repurposing16. However, while theoretically sound, this approach is impractical for drug repurposing, as discussed earlier. Our drug-target enrichment analysis showed that such a direct application of structural controllability was no better than random selection . Many druggable control hubs have immunity and antiviral functions such as regulating apoptotic signaling, cellular response to stress, leukocyte proliferation, and cell population proliferation , S4. Nutral RdRp , 3B. RIP48 and the data of a preliminary clinical trial of critically ill patients49. Altogether, the biological functions and experimental data suggested that the drug prevents exaggerated autoinflammatory immune responses68,69 and alleviates the burden of cytokine storms69 in severe Covid-19 cases.An interesting result is identifying Fostamatinib as a Covid-19 drug, particularly suitable for hospitalized patients , S5. ThiA substantial number of control hubs in the 2-step community of the human PPI network are not targets of any existing drug. These control hubs, particularly the membrane proteins that function on the NF-κB pathway , can be used to propose testable hypotheses for new drug development for Covid-19 therapy.In summary, our study presents a novel approach to drug repurposing that has significant implications for COVID-19 treatment and beyond. By focusing on control hubs as drug targets, we can potentially unlock a new strategy for combating not only current but also future viral threats. Furthermore, many of the control hubs we identified as not yet targeted by existing drugs present exciting avenues for the development of new antiviral medications.The new method consists of the following four major steps discussed in the subsequent subsections.Construction of a biological network. In the current study, an integrated network of human PPI, virus PPI, drug targets, and drugs;31; the algorithmic details are in Supplemental Method S3.Identification of control hubsDetermination of the Assessment and validation of the new method by comparison with nine existing gene selection methods, including the structural-controllability-based driver-node method.25. This is the largest homogenous human protein interactome with data collected primarily from HEK923T cells and validated in multiple orthogonal assays. The network consists of 9,092 nodes or proteins and 64,006 interactions (Table S1).The central layer of the network contained the human protein-protein interaction (PPI) network that was constructed using the human Huri-Union binary protein interaction dataset70.The SARS-CoV-2 AP-MS data26 from HEK293T cells was added to include the layer of viral proteins. The dataset contains 332 high-confidence virus-host interactions between 27 SARS-CoV-2 proteins and 332 human proteins, which were used to link the human and virus PPI subnetworks. Since the human PPI network contains only 9,092 proteins, the final triple-layer network contains 169 interactions between 22 viral and 169 human proteins (Table S2). The 3D Structural Interactome between SARS-CoV-2 and host proteins was retrieved from SARS-CoV-2-Human Interactome Browser28. The links between drugs and their protein targets were used to link the human PPI subnetwork and drug subnetwork. We only included drugs approved by FDA and under investigation. The drug-target interactome contains 17,780 interactions between 2,981 drugs and 2,914 target proteins (Table S3). The information on drug categories in DrugBank was used to group drugs (Table S5).The network was further expanded to include the layer of drugs and their human protein targets using the data from DrugBankdriver nodes29 , three related to network shortest paths , three related to network structures , and a classical web ranking algorithm (page rank74). A detailed description of these ranking algorithms is in networkx75 and Supplemental Method S1.Nine popular node ranking methods were used to compare with the new control-hub-based method. These include two methods related to node degree  pathway annotation and Gene Ontology (GO) annotation were performed using Metascape


As we enter the fourth year of the coronavirus disease 2019 (COVID-19) pandemic, it has become obvious that adult survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are susceptible to numerous complications in various organ systems. SARS-CoV-2 placental infection is an unanticipated complication of COVID-19 during pregnancy. We hypothesize that fetal survivors of SARS-CoV-2 placentitis are susceptible to long-term cardiovascular complications. BackgroundAs we recently passed the three-year mark with the pandemic, we are beginning to see the effects that coronavirus disease 2019 (COVID-19) has caused on a global spectrum . In addiHypothesisThe effects of the SARS-CoV-2-infected placenta will be detrimental to the surviving fetus later in life. Intrauterine exposure to maternal disease processes, such as cyanotic heart disease, heart failure, and pulmonary hypertension, has been shown to affect the future cardiovascular health of the fetus. Intrauterine asphyxia is also well known to have long-term effects on the fetus. We propose that SARS-CoV-2 infection of the placenta via the mother and the maternal effects of COVID-19 have similar long-term effects on the surviving fetus. The severity of the placental infection likely will also play a role.Support for the hypothesisFetuses who survive intrauterine chronic fetal hypoxia or acute fetal hypoxia have an increased risk of detrimental effects that may manifest in the pediatric or adult time period. We propose that survivors of SARS-CoV-2-infected pregnancies are likely to have long-term effects in adulthood.Chronic Fetal HypoxiaChronic fetal hypoxia resulting from maternal conditions has been shown to have effects on the neonate such as poor growth . In knowInfants who survive maternal chronic hypoxic disease during pregnancy have been shown to be growth-restricted, which is considered a risk factor for cardiovascular disease. In adulthood, these surviving fetuses have an increased risk of arterial hypertension, increased systolic blood pressure, premature coronary heart disease, stroke, and diabetes. There have been similar findings in animal models of chronic intrauterine hypoxia .The mechanism of these long-term cardiovascular changes due to intrauterine hypoxia is likely epigenetic changes in myocardial and vascular development. Prenatal programming likely leads to long-term changes in the expression of numerous factors , which are responsible for the regulation of cardiac and vascular function .In addition, cardiac myocyte response to chronic intrauterine hypoxia, such as apoptosis or exiting the cell cycle, likely contributes to fewer but hypertrophied cardiac myocytes. Although this is more energy efficient in the fetus, it likely results in less compliant ventricles, which lead to cardiovascular disease in adulthood .Acute Fetal HypoxiaSevere acute hypoxia is well known to have detrimental effects on the fetus. Adaptive cardiovascular responses aim to preserve blood flow to the brain and heart at the expense of other organs. In a sheep animal model of acute intrauterine asphyxia, it has been shown that the surviving fetuses can have brain injury due to disruption of blood flow. The mechanisms also include oxidative stress responses that lead to deficiencies in nitric oxide pathways which may damage the cardiovascular system. Despite advances in care, survivors of acute intrauterine asphyxia frequently have significant brain injury  .Hypoxia and acidosis from acute intrauterine asphyxia contribute to postnatal pulmonary hypertension. In the developing fetus, the right ventricle is dominant and responsible for moving blood into the high-pressure pulmonary artery. In fetuses exposed to hypoxic environments, the right ventricle is at risk of damage; cardiac dysfunction and pulmonary hypertension would be expected to occur postnatally. This can then lead to poor pulmonary blood flow, ventilation-perfusion mismatching, decreased preload of the systemic circulation, and the resulting reduction in cardiac output. These cardiovascular changes also contribute to the development of hypoxic-ischemic encephalopathy and may ultimately result in hemodynamic instability .Effects of Chronic and Acute Hypoxia on Transitional CirculationThe cardiovascular conditions mentioned above may result in fetal growth restriction (FGR), which is generally defined as a birth weight less than the 10th percentile when controlled for gestation and sex . ChangesIt appears that FGR is not common in SARS-CoV-2-infected mothers. In 2022, Wilkinson et al. conducted an analysis of over 36,000 births and did not find significant data that would confirm that FGR is a common result of maternal infection . HoweverAnimal models of acute and chronic fetal hypoxia on transitional circulation have helped elucidate the mechanisms that likely lead to long-term detrimental effects in survivors. These changes involve persistent changes in cardiac function and vascular remodeling . StudiesPathophysiologic Mechanism of Long-Term Cardiovascular Changes in Fetal HypoxiaFetal distress has been reported in nearly 50% of all births in mothers infected with SARS (CoV-1 or CoV-2) . In othePatent Ductus ArteriosusThe patent ductus arteriosus (PDA) in the developing fetus allows oxygenated blood from the placenta to bypass the pulmonary circulation and enter the systemic circulation via the descending aorta. This patency is maintained by the presence of high blood flow along with circulating prostaglandins from the placenta . After dIt is abnormal to observe post-natal patency in the ductus arteriosus; this can be seen in many pathologic conditions . The conPost-natal complications of PDA can include respiratory distress syndrome and pulmonary hypertension . It is pVaccinated vs. Unvaccinated MothersThe two-dose regimen of the COVID-19 vaccine leads to the production of immunoglobulin gamma (IgG) antibodies against the virus in the mother’s immune system. It has also been documented that through the utilization of IgG receptors in the placenta, these antibodies are able to traverse the placenta and confer up to six months of protection postpartum to the fetus/infant. One study documented that out of 379 infants hospitalized due to COVID-19 infection, 88% of them were born to unvaccinated mothers. Fifteen percent of that 88% received mechanical ventilation, vasoactive infusion, and/or ECMO, and one infant died . Other sEvaluation of hypothesisTo assess the effects of SARS-CoV-2 placental infection on the surviving fetus, we propose long-term monitoring throughout childhood and adulthood for these patients. This study should include an adequate number of survivors; these numbers are easily reached during the various COVID-19 surges. Evaluation should include studies of cardiac function such as echocardiogram and blood pressure measurements. It is important to consider the selection of controls for these studies. The time of the pandemic was extraordinarily stressful for all people, so there may be stress effects in fetuses born to non-COVID-19-infected mothers during the pandemic. Controls should be selected using gestational age and sex-matched controls who were born on the same day to non-COVID-19-affected mothers in the same hospital.ConclusionSARS-CoV-2 placentitis resulting from maternal infection during pregnancy likely has long-lasting effects on survivors. Prior to the pandemic, numerous studies have shown long-term cardiovascular effects in survivors of acute perinatal asphyxia and chronic intra-uterine hypoxia (infants with FGR). The underlying mechanisms that lead to long-term damage likely result from the effects of acute and chronic hypoxia on the pre-natal and peri-natal time periods. These changes are likely to affect survivors of fetal SARS-CoV-2 placentitis well into adulthood.


BackgroundPhysical fitness is of utmost importance to athletes as it ensures better performance in competitive sports. Athletes who contracted COVID-19 frequently experienced persistent symptoms for weeks or months afterward. Due to the direct effects of COVID-19 infection on pulmonary, cardiovascular, and neurological systems, combined with the negative effects of isolation and inactivity, it has been observed that physical fitness decreases in individuals. This study aimed to evaluate the physical fitness of young athletes in the age group of 20 to 30 years after mild-to-moderate COVID-19 infection and compare them with unaffected athletes of the same age group.MethodologyA field-based, cross-sectional, comparative study was conducted from July 2022 to August 2022 in Nagpur, India. Physical fitness levels of 50 young athletes in the age group of 20-30 years who never got infected with COVID-19 were compared to 50 athletes with a recent history of mild-to-moderate COVID-19 infection using the Harvard step test, breath-holding test, and peak expiratory flow rate measurement. Participants were included based on COVID-19 diagnosis using standard procedures and confirmation of recovery through negative reverse transcriptase polymerase chain reaction tests.ResultsOverall physical fitness of athletes who suffered from mild-to-moderate COVID-19 infection was significantly less than those who were not infected. Compared to their non-COVID-19 counterparts, the COVID-19-recovered athletes showed reduced physical fitness index , reduced peak expiratory flow rate , and reduced breath-holding time .ConclusionsCOVID-19 had a significant impact on various components of physical fitness which may potentially affect the athletic performance and overall well-being of young athletes. The COVID-19 pandemic has not only posed a threat to public health but has also disrupted various aspects of society, including sports and physical activities. Young athletes, who are essential contributors to the sporting world, have been affected by the virus and its associated consequences. There are now consistent reports of athletes reporting persistent and residual symptoms many weeks to months after COVID-19 infection [The main site of COVID-19 infection is the lungs, as the virus gets into the body through the respiratory pathway. The infection starts spreading in one lung and progressively into another leading to impairment of the lungs to swap oxygen and carbon dioxide . PhysicaThe purpose of this pilot study was to investigate the effects of COVID-19 infection on the physical fitness of young athletes aged 20 to 30 years. By examining the various components of physical fitness, this study aimed to provide preliminary insights into the potential long-term impact of COVID-19 on the athletic performance and overall well-being of this population.This pilot study employed a cross-sectional design and was conducted over a two-month period from July 2022 to September 2022. The research was conducted at two locations: the Department of Physiology, Government Medical College, Nagpur, India, and the Ishwar Deshmukh College of Physical Education Practice Ground, Hanuman Nagar, Nagpur, India. The study enrolled a total of 50 young athletes , aged 20 to 30 years, who had previously recovered from mild-to-moderate COVID-19 infections, verified through standard diagnostic procedures. An additional 50 athletes , who had never contracted COVID-19, were selected as the control group for comparative analysis. Participants in both categories (cases and controls) were recruited using a convenient sampling method. The criteria for recovery from COVID-19 included the cessation of self-isolation for seven days and the confirmation of negative laboratory results via reverse transcriptase polymerase chain reaction (RT-PCR) testing. Accordingly, physical fitness was tested after seven days of isolation and a concurrent negative COVID-19 report.The study included athletes who met the following inclusion criteria: individuals willing to participate, those aged between 20 and 30 of any gender, those who tested positive for COVID-19 via RT-PCR (cases), those who were never COVID-19 positive on RT-PCR testing (controls), and individuals engaging in regular physical exercise for the past three to five years. Exclusion criteria comprised individuals who were unwilling to participate; those not consistent with physical exercise; individuals with any physical or psychological illnesses; a history of smoking, alcohol, or drug consumption; and those with chronic illnesses.Sample size calculation2 p (100-p) / d2 = 4pq/d2. Using the formula, the sample size for our study was calculated as 32. We considered a precision level of 10% and a prevalence of 10% for COVID-19 infection among athletes. Accordingly, we recruited 50 cases and 50 controls in the study.The sample size was calculated using Cochran’s formula as follows: n = ZMeasurementsA comprehensive assessment of physical fitness components was conducted on each participant in the morning after light breakfast. The physical fitness components are discussed below.Harvard Step TestIn 1943, the Harvard step test was developed to assess the aerobic fitness and development of an athlete’s cardiovascular system . The tesPeak Expiratory Flow Rate MeasurementPeak expiratory flow rate (PEFR) is the volume of air forcefully expelled from the lungs in one quick exhalation. It is a reliable indicator of ventilation adequacy as well as flow obstruction . The tesBreath-Holding TestThis test is used as a rough index of the cardiopulmonary reserve, measured by the length of time a person can hold his or her breath . AthleteStatistical analysisGraphPad Prism 7.0 was used for data analysis. The data were expressed as mean and standard deviation (mean ± SD) and percentage (n%) in Microsoft Excel 2010. Data were tested using the Shapiro-Wilk test and were found to be normally distributed, and the sample size was above 30 to consider the data as normal. Between groups, the PFI, PEFR, and breath-holding time (BHT) were compared using the unpaired t-test as our data were normal in distribution [Ethical considerationsThe study was approved by the Institutional Ethics Committee of the Government Medical College, Nagpur (reference ID: 2022-01498/EC/Pharmac/GMC/Nagpur). The study objectives were explained to participants in their mother tongue and their questions were answered. Only participants who signed an informed consent form were enrolled. Participation in the study was strictly voluntary and participants were free to decline to answer any question or totally withdraw if they so wished at any time.The study conducted on a group of 50 young athletes, comprising 31 males and 19 females who had previously recovered from COVID-19, yielded preliminary but significant findings. These findings suggest that COVID-19 infection has a substantial impact on the physical fitness of these athletes.The data, illustrated in Figures Assessment of physical fitness does not always require heavy or expensive machinery, physical fitness can also be effectively assessed by some simple parameters and devices, such as those used in this study.Harvard step test assesses the aerobic fitness and cardiovascular system of an individual. In males, a fitness level above 115 is considered excellent, and in females, a level above 91 is considered excellent . In our PEFR is a significant parameter for testing ventilation adequacy. Assessment of PEFR is important for the early detection and prevention of chronic obstructive pulmonary disease . Males hBHT involves holding of breath for the maximum possible time, and this duration is an indication of the cardiopulmonary reserve of an individual. An average human can hold their breath for 30 seconds, while athletes have the ability to hold their breath for more time, which indicates their huge cardiopulmonary reserve which plays a key role in performing physical activities . ResultsAs can be seen, the negative effects of COVID-19 infection on the pulmonary, cardiovascular, and neurological systems and the restriction of physical activity due to the pandemic led to a decrease in some physical fitness parameters . PrelimiThis pilot study provides initial insights into the effects of COVID-19 infection on the physical fitness of young athletes aged 20 to 30 years. The findings suggest that COVID-19 had a significant impact on various components of physical fitness, including cardiopulmonary efficiency, ventilation adequacy, and cardiopulmonary reserve. These observed changes may potentially affect the athletic performance and overall well-being of young athletes.In conclusion, this pilot study highlights the need for further investigations into the impact of COVID-19 on physical fitness in young athletes. Understanding the long-term effects of the virus on athletes’ physical abilities and designing appropriate interventions will be crucial for their successful recovery and return to sports. By addressing these issues, we can better support the physical and mental well-being of young athletes in the post-pandemic era.


The emergence of multidrug-resistant NDM-1-producing enterobacteriaceae strains has become a threat to inpatients, especially to immunosuppressed ones, such as kidney transplant recipients. NDM-1 is a carbapenemase that makes gram-negative bacteria resistant to many types of antibiotics. The incidence of carbapenemase-producing enterobacteria infection in solid organ transplant recipients is around 3 to 10%, with a mortality rate of up to 30%.We present a case series of 4 patients with NDM-1-producing enterobacteria isolated in urine cultures or rectal swabs. We also conducted a cross-sectional study 30 days after patient identification, collecting surveillance cultures  from all inpatients to assess the extent of spread of this resistance mechanism; a total of 101 patients were included.Two patients were adequately treated with negative control cultures. The other two patients were not treated because they were asymptomatic and had subsequent negative urine cultures. No new colonization was identified in the cross-sectional screening, and no new cases of urinary NDM-1 infection were recorded after a 4-year follow-up.Surveillance for infections caused by multidrug-resistant strains in hospitals treating immunosuppressed patients should be continued and prompt action should be taken in cases of outbreaks of multidrug-resistant infections. The identification of carbapenem-resistant enterobacteria by the production of the enzyme KPC was first performed in our institution in 2009, but no cases of resistance by the production of the New Delhi Metallo-beta-lactamase (NDM) enzyme have been identified yet. Here, we report 4 cases of NDM-producing carbapenem-resistant enterobacterial strains identified in urine or rectal swab cultures from kidney transplant recipients at our center. We also conducted a cross-sectional surveillance 30 days after the hospital outbreak by rectal swab culture from all inpatients to assess the spread of this multidrug-resistant bacteria, totaling 101 examined patients. This project was approved by the local ethics committee (CAAE 15815419.7.0000.5505).th postoperative period, he presented dysuria, and urine culture was collected for investigation. Carbapenem-resistant Klebsiella pneumoniae (bla KPC-positive PCR) was isolated and the patient received meropenem associated with amikacin for 7 days, being discharged at the 20th postoperative period. After 11 days, he developed graft dysfunction (creatinine of 1.89 to 2.61 mg/dL) with a new carbapenem-resistant Klebsiella pneumoniae isolation with bla KPC and bla NDM-positive PCR. He received endovenous amikacin and meropenem, but according to the sensitivity profile of the culture, meropenem was changed to oral phosphomycin . Nine days after hospital discharge, she presented dysuria and mild renal dysfunction (creatinine of 1.46 mg/dL). Ciprofloxacin was started empirically. A few days later, urine culture showed growth of carbapenem-resistant bla NDM , evolvinKlebsiella pneumoniae. A surveillance rectal swab was collected and resulted in the isolation of the bacteria with PCR positive for bla NDM. However, as the patient was asymptomatic, the medical team decided not to prescribe any treatment. Kidney function was stable, and the patient was discharged without complications.Case 3 - A 29-year-old man who had been in hemodialysis for 3.5 years at a clinic 20 km from Hospital do Rim underwent a kidney transplantation from a living donor. Four days after surgery, the patient had an accidental contact with a patient colonized by carbapenem-resistant Enterobacter aerogenes was isolated with positive PCR for bla NDM. Treatment with oral phosphomycin and intravenous meropenem for 14 days was prescribed. The symptoms improved and urine culture after treatment was negative. The patient remained hospitalized due to disseminated cryptococcosis and his clinical condition continued to worsen despite appropriate treatment for urinary tract infection. The patient required intensive care, but died 60 days after hospitalization due to the disseminated cryptococcosis.Case 4 - A 38-year-old man, who underwent a kidney retransplant from a deceased donor seven years earlier was being treated for disseminated cryptococcosis with oral fluconazole when he was hospitalized for investigation of consumptive syndrome and fever. After six days of hospitalization, as the patient continued to have fever, a urine culture was collected from which None of the four cases described above were in the same inpatient unit during hospitalization.A cross-sectional analysis was performed in all inpatients 30 days after the identification of the last case to assess the NDM-producing enterobacterial dissemination in the hospital. We collected surveillance rectal swabs from 101 inpatients, and no new cases were detected. In addition, after 4 years of follow-up, no new diagnosis of infection with an NDM-1 producing multidrug-resistant strain was made in urine or rectal swab cultures collected at our center.,,Preventing the spread of multidrug-resistant bacteria is an important issue worldwide. Detection of the NDM-1 enzyme-producing bacteria is a major new challenge for infectologists due to their great potential for spread and few therapeutic options. Due to its broad-spectrum drug resistance, NDM-1 strains are only susceptible to polymyxin, aminoglycosides, or the combination of two carbapenems with phosphomycin-The incidence of carbapenemase-producing enterobacteria infection in solid organ transplant recipients is 3 to 10%, with a mortality rate reaching 30%,,The bactericidal activity of phosphomycin involves the inhibition of proteoglycan synthesis of the bacterial cell wall, increasing cell membrane permeability of gram-positive and gram-negative bacteriaOf the 4 patients reported with NDM-1 positive culture, two of them developed an infection and were adequately treated with negative control cultures, and the other two patients were not treated because they were asymptomatic and also had subsequent negative urine cultures. No new colonization was identified in the cross-sectional screening in 101 inpatients, and no new cases of urinary NDM-1 infection were recorded at the hospital after a 4-year follow-up.In this context, NDM-1 strains are particularly dangerous for several reasons: (a) most plasmids detected in these bacteria are transferable and capable of broad rearrangement, suggesting widespread horizontal transmission and flexibility among bacterial populations; (b) there is a lack of a standardized and widely accepted phenotypic test for routine detection of metallo-beta-lactamases (MBLs); (c) there is likely to be a high prevalence of undetected asymptomatic carriers, particularly in patients with chronic diseases who are frequently in the hospital setting; (d) there is a lack of effective, toxicity-favorable antibiotics for the treatment of multi-drug resistant NDM-1-expressing bacteria.In addition, antibiotic resistance is spread by the misuse of antibiotics and by inadequate infection prevention and control. However, some measures could be taken to contain the spread of multidrug-resistance bacteria at hospital level, as recommended by the World Health Organization (WHO)Surveillance and control of infections caused by multidrug-resistant strains in hospitals, especially those treating immunosuppressed patients, should be continuous and rigorous so that actions can be promptly taken to control multidrug-resistant bacteria outbreaks when they occur. The cases described here shed light to the high exposure to multidrug-resistant bacteria in hospitals specialized in solid organ transplantation, which receive recipients from different regions of the country, most of them on hemodialysis. In addition, these hospitals receive organs taken from deceased donors hospitalized in intensive care units, often for extended periods. Finally, according to the current knowledge, the use of phosphomycin alone or preferably in combination with non-nephrotoxic antimicrobial agents should be the therapy of choice for NDM-1 infections in solid organ recipients.


Long-acting injectable antipsychotics (LAIA) are used in diagnoses other than schizophrenia. Over the last two decades, LAIAs have been developed with less administration frequency, going from 2-weekly presentations to 6-monthly presentations. The 6-monthly paliperidone palmitate has recently been released, allowing a reduction in the frequency of administration compared to the 1-monthly presentation and the 3-monthly presentation. Descriptive studies based on real clinical evidence can be very useful to assess clinical outcomes.The main objective of the study is to describe the use of 6-monthly paliperidone palmitate in patients with schzophrenia, providing variables that objectify the evolution such as the number of psychotic decompensations.Retrospective descriptive study with a sample selected by non-probabilistic consecutive sampling, retrospective type, in a time interval of 10 month (n=80). The patients selected were all those who received 6-monthly paliperidone palmitate treatment from after 10 months of use at Hospital Universitario Infanta Elena. A descriptive analysis was performed. Mean and standard deviation were calculated for quantitative variables and N and percentage for categorical variables.A total of 80 administrations of 6-monthly paliperidone palmitate were performed in the study. None of the patients presented adverse reactions related to the administration of the drug, not reporting local pain or inflammation of the puncture area, except for the characteristic discomfort of an intramuscular puncture. Regarding the efficacy of 6-monthly paliperidone palmitate, none of the patients presented a psychotic decompensation after its administration, maintaining psychopathological stability after the change. The switch to 6-monthly paliperidone palmitate was made from both 1-monthly paliperidone palmitate and 3-monthly paliperidone palmitate, both showing the same efficacy. Regarding tolerability, all the patients who were administered 6-monthly paliperidone palmitate were previously treated with the monthly and quarterly presentation of the same molecule, having presented good tolerability to it, maintaining said tolerability after treatment. change to 6-monthly paliperidone palmitate, with no adverse reaction being recorded after the change. The adherence presented by the patients was very good, performing 100% of the administrations.6-monthly paliperidone palmitate may be an effective and well-tolerated treatment for the treatment of schizophrenia and other diagnoses such as bipolar disorder or borderline personality disorder. According to objective data, 6-monthly paliperidone palmitate could be an effective and well-tolerated treatment as an alternative to monthly and quarterly presentations of the same molecule. Longitudinal studies must be carried out to confirm this hypothesis.None Declared