Can an informatics tool improve detection of antibiotic allergic-type events?In this cohort study of 36 344 patients who underwent cardiovascular implantable electronic device procedures, an electronic algorithm with high positive predictive value was developed that can be used to measure the occurrence of periprocedural antibiotic allergic-type events in near real-time. Most antibiotic allergic-type reactions were moderate in severity.The algorithm developed in this study could be used to detect when antibiotic allergic-type reactions occur to provide direct clinician feedback about harms. This cohort study describes the development of an informatics tool to improve detection of periprocedural antibiotic allergy adverse events. Standardized processes for identifying when allergic-type reactions occur and linking reactions to drug exposures are limited.To develop an informatics tool to improve detection of antibiotic allergic-type events.This retrospective cohort study was conducted from October 1, 2015, to September 30, 2019, with data analyzed between July 1, 2021, and January 31, 2022. The study was conducted across Veteran Affairs hospitals among patients who underwent cardiovascular implantable electronic device (CIED) procedures and received periprocedural antibiotic prophylaxis. The cohort was split into training and test cohorts, and cases were manually reviewed to determine presence of allergic-type reaction and its severity. Variables potentially indicative of allergic-type reactions were selected a priori and included allergies entered in the Veteran Affair’s Allergy Reaction Tracking (ART) system , allergy diagnosis codes, medications administered to treat allergic reactions, and text searches of clinical notes for keywords and phrases indicative of a potential allergic-type reaction. A model to detect allergic-type reaction events was iteratively developed on the training cohort and then applied to the test cohort. Algorithm test characteristics were assessed.Preprocedural and postprocedural prophylactic antibiotic administration.Antibiotic allergic-type reactions.The cohort of 36 344 patients included 34 703 CIED procedures with antibiotic exposures ; median duration of postprocedural prophylaxis was 4 days . The final algorithm included 7 variables: entries in the Veteran Affair’s hospitals ART, either historic  or observed ; PheCodes for “symptoms affecting skin” , “urticaria” , and “allergy or adverse event to an antibiotic” ; keyword detection in clinical notes ; and antihistamine administration alone or in combination . In the final model, antibiotic allergic-type reactions were identified with an estimated probability of 30% or more; positive predictive value was 61% ; and sensitivity was 87% .In this retrospective cohort study of patients receiving periprocedural antibiotic prophylaxis, an algorithm with a high sensitivity to detect incident antibiotic allergic-type reactions that can be used to provide clinician feedback about antibiotic harms from unnecessarily prolonged antibiotic exposures was developed. Clostridioides difficile infection, acute kidney failure, and immunologically mediated hypersensitivity reactions.3 Antibiotics are among the most common drugs associated with hypersensitivity reactions in hospitalized patients.4 The downstream effects posed by an antibiotic allergy include increased length of stay and health care costs, use of broad-spectrum antibiotics, and mortality.7Adverse events associated with antibiotic use include 9 Despite clinical guidelines recommending against extending duration of prophylaxis following skin closure, prolonged postprocedural antibiotics are commonly prescribed in some clinical settings, increasing the risk of adverse events, including antibiotic allergic-type reactions.13 However, the rate of antibiotic-related adverse reactions and allergy events that occur in this context is unknown.Antibiotics that most commonly cause hypersensitivity reactions  are the same drugs commonly prescribed for surgical site infection prophylaxis.There is no current process for antibiotic drug reaction surveillance. Lack of adverse event detection prevents feedback to clinicians about direct patient harms of inappropriate antibiotic use. Thus, the aims of this retrospective, multicenter national cohort study were (1) to develop an electronic tool using structured and unstructured data elements available within the Veterans Affairs (VA) electronic health record (EHR) to identify incident antibiotic allergic-type reactions and (2) to characterize the nature and severity of these reactions.STROBE) reporting guidelines for cohort studies.The VA Boston Healthcare System institutional review board approved this as an exempt study with a waiver of informed consent given the retrospective and observational nature of the study. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology  procedures  performed at Veterans Health Administration (VHA) facilities from October 1, 2015, to September 30, 2019. Data were analyzed between August 1, 2021, and January 31, 2022. Individual CIED procedures were included in the study if they had received 1 or more antibiotic(s) within 1 day before or after their procedure.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision [ICD-10] diagnosis codes, procedure codes, patient characteristics), structured tables (orders and dispensed medications), and unstructured clinical text notes. Duration of antibiotic exposure following CIED procedure was calculated based on the number of days of documented antibiotic administrations (inpatient data) and/or the number of days of antibiotic fills (outpatient exposures). Patient comorbidities were extracted using the Agency for Healthcare Research and Quality Elixhauser Comorbidity Software.17EHR data were extracted from the VA Corporate Data Warehouse using SQL Server Management Studio 18 (Microsoft Corp). Data sources accessed include administrative files  system , allergy diagnosis codes , medications administered to treat allergic-type reactions , text searches of clinical notes for keywords and phrases indicative of a potential allergic-type reaction, and consults to allergy and dermatology  and the subsequent 45-day window period was completed. The surveillance period for antibiotic-associated allergic-type reactions was a maximum of 10 days after the last date of antibiotic exposure, which was calculated based on medication administration data, days of antibiotic dispensed, or both. Manually abstracted data included procedure date, adverse event date and type , triggered variable, culprit antibiotic, reaction noted, treatment received, and change in level of care . Each event was then classified into allergic-type reaction, adverse reaction, or other or no event occurrence. Allergic-type reactions were defined as allergic signs or symptoms that could be immune-mediated hypersensitivity reactions, such as rash, hives, or anaphylaxis occurring after antibiotic exposure.2 Adverse reaction was defined as a nonimmunologically mediated undesired response to an antibiotic .2 Among patients who experienced an allergic-type reaction, severity of allergies was classified as mild, defined as not requiring medication or change in level of care; moderate, defined as requiring medication but no change in level of care; and severe, defined as requiring medication and change in level of care or intubation.25The manual medical record review process included a standardized review tool.26 rather than ICD-10 codes  were selected using a generalized linear model with a logit link (via the SAS Procedure HPGENSELECT), with a least absolute shrinkage and selection operator (LASSO) regression analysis for variable selection (SAS Enterprise Guide version 8.2 [SAS Institute]). Potential factors were iteratively updated to increase estimated probability of identifying true antibiotic allergic-type reactions, eg, by adjusting keyword searches and using diagnosis phenotype codes (PheCodes)Different iterations of the model were subsequently tested using logistic regression to estimate odds ratios (ORs) for different variables and estimate the probability of an antibiotic allergic-type reaction. The final version of the model was selected based on the estimated probability cutoff with the highest positive predictive value (PPV) and C statistic. Unadjusted and adjusted (full model) ORs for the outcome and the receiver operating characteristic were calculated in the training data set using the final model.27After selecting a predictive model using the training medical record review sample, logistic regression coefficients were applied to the testing cohort to obtain estimated probabilities that an allergic-type reaction had occurred. The test medical record review sample was selected according to a predetermined but arbitrarily selected estimated probability distribution to oversample high-probability cases. After reviewing the sampled procedures from the testing cohort, we assessed criterion validity  of the final model and compared it with the model performance in the training medical record review sample. Criterion validity of the algorithm was assessed with criterion-standard manual medical record review.26A total of 36 344 patients underwent CIED procedures during the study period, and 34 703 (96%) had a periprocedural antibiotic exposure and were included in the study. The median duration of postprocedural antibiotic prophylaxis in the whole cohort was 4 days . The total population was divided into a training data set (17 350 participants) and a testing data set (17 353 participants). Most included patients were male (34 008 [98%]) and White (26 576 [77%]). The mean (SD) age was 72 (10) years .21,22,26ICD-10 code; and PheCodes for urticaria, adverse event to antibiotic and penicillin, and rash and skin eruption . In the training sample, the initial set of variables selected by the LASSO included observed ART flag; keywords documented in clinical notes; corticosteroid receipt; new eruption . PrespecThe final model included 7 variables indicative of an antibiotic allergic-type reaction during the 45-day period following antibiotic exposure  . The varThe logistic regression model from the training cohort was applied to the testing cohort of 17 353 participants. A total of 17 259 cases (99%) fell under the PPV threshold for detecting potential allergic-type reactions; 120 cases underwent manual review . In this sample, 87% of allergic-type reactions (27 cases) fell into the high probability category. The PPV of the final model in the test data set was 61% , and sensitivity was 87% . The ART flag alone had lower sensitivity but similar PPV as the more complex model .25 Among cases with allergic-type reactions, the median (IQR) duration of postprocedure exposure was 5 (2-7) days; the same time frame when most events (75 [72%]) occurred. The most frequently administered antibiotics associated with allergic-type reactions included cephalosporins (40%) and vancomycin (23%), the same antibiotics most frequently administered for periprocedural prophylaxis.Allergic-type reactions by class of antibiotic, intervention administered, and severity classification are presented in Among the manually reviewed cases from the whole cohort, 104 antibiotic allergic-type reactions occurred following 102 exposures . Of these, 96 were due to antibiotics administered for periprocedural prophylaxis, and 81 allergic-type reactions (84%) were attributed to antibiotics administered for longer than 24 hours following skin closure. Of the 96 antibiotic allergic-type reactions attributed to periprocedural prophylaxis, 60 (63%) had an observed ART flag, 23 (24%) had a historic ART flag, and 30 of 96 cases (38%) did not have the allergy listed in the electronic medical record.Most antibiotic allergic-type reactions detected in the inpatient setting and were moderate in severity, requiring administration of antihistamine, corticosteroid, or intravenous epinephrine or a combination but did not require change in level of care. Most cases that required change in level of care  were outpatients treated in the emergency department. Intubation or intensive care unit was not required for any of the reactions with cephalosporins or vancomycin. One patient who presented with pneumonia and received azithromycin developed anaphylactic shock, was intubated in the emergency department, and was admitted into the intensive care unit.7 Despite this, guideline-discordant prolonged prophylaxis lasting longer than 24 hours after skin closure remains common in some surgical and procedural settings, including the cardiac electrophysiology laboratory.28 Identification of allergic-type reactions in near real-time creates an opportunity for audit and feedback to clinicians about patient harms of antibiotic prescribing and an opportunity to encourage adoption of evidence-based practices regarding antibiotic administration for postprocedural prophylaxis. Systematic surveillance may be particularly important in outpatient and procedural settings, as many events may occur after discharge and thus clinicians remain unaware of preventable harm caused by inappropriate antibiotic administration. The algorithm developed and validated in this study helps close this gap and can potentially be used to enhance identification of allergic-type reactions and improve quality and safety of medical care.Antibiotics are one of the most common causes of medication adverse events.16 These findings highlight the importance of perioperative antibiotic stewardship to limit harms of unnecessary antibiotic exposures and the need for innovative strategies to provide audit and feedback to clinicians about patient harm caused by unnecessary antibiotic exposures.Most allergic-type reactions occurred shortly after antibiotic exposure  and were attributed to antibiotics administered for guideline-discordant, prolonged prophylaxis.30 The higher rate in our study is likely due to a combination of the duration of prophylaxis and exposure to different agents for prophylaxis . Importantly, given these procedures were performed among individuals without an underlying infection, we were able to identify allergic-type reactions without the need to distinguish allergic reactions from other entities that cause rash, such as viral exanthems.In our study, the rate of antibiotic allergic-type reactions occurring with prolonged postprocedural antibiotic use was higher than previously reported rates (0.9% for cefazolin and 1.1% for vancomycin).C difficile infection following a single exposure) are rare; however, when a broader array of outcomes is considered, incidence of overall harm to the individual patient increases and contributes more substantially to adverse patient outcomes. More research is needed to develop antibiotic-use harm metrics that fully capture all harms caused by antibiotics to better inform and adapt clinical practice.This study adds to previous literature about the harms of extending prophylaxis beyond the guideline-recommended period and can be used in future investigations to consider a composite adverse event risk metric from antibiotic exposure, rather than considering each harm individually. Antibiotic-associated adverse events when considered individually  fell below the probability threshold that would require manual adjudication. Ideally, the algorithm would assist with attributing an allergic-type reaction to a specific exposure. However, in our cohort, patients received more than 1 antibiotic during the specified period, and pinpointing the causative agent was not possible without manual adjudication.8 Mild reactions that did not require medical intervention are less likely to be detected, as they are less likely to require medical care and therefore documentation in the EHR. However, they are likely less important from a clinical standpoint. Severe reactions were also less likely to be identified, likely because they occur less frequently.31 However, it is possible that severe reactions are more common than was identified, as patients with severe reactions may be taken to the nearest emergency department, not necessarily to a VA medical center, and therefore, the events might not be documented in the VA EHR.The algorithm primarily detected allergic-type reactions of moderate severity. This may be due to moderate severity allergic-type reactions being more common, or due to inherent features of the algorithm. Cutaneous manifestations were the most commonly reported allergic-type reactions, and these are easily treated in the inpatient setting with diphenhydramine or steroids, which were included in the algorithm.ICD-10-DM codes  were copied forward in EHR documentation, even though no penicillin exposure occurred during the incident admission. Missing data due to inability to detect adverse reactions that are treated outside the VA is another limitation and may have affected the number of cases identified and the true severity distribution of antibiotic allergic-type reactions. Our validation method focused on oversampling high probability cases, which may have led to an underestimation of the true incidence of adverse events due to limited review of potentially false-negative cases. It is not clear how well the tool would perform outside of the closed VA health care system or other EHRs or applied to other clinical settings or more complex or prolonged surgical procedures.Our study is subject to the limitations of documentation in the EHR and sampling methodology. False-positive variable detection related to historic 32; thus, the absolute incidence of allergic-type reactions may be underestimated in this population relative to populations with more women. Additionally, manual review was completed on a relatively small sample of the total cohort, and by a single adjudicator.Another limitation is that this study was conducted within the VA health care system, and the cohort included predominantly older White male patients. Drug allergies are much more commonly reported in female than male patientsIn this cohort study, we developed and validated an informatics tool using 7 variables composed of structured and unstructured data for the detection of antibiotic allergic-type reactions. This model had a good PPV for detecting antibiotic allergic-type reactions and can be operationalized to provide clinicians with real-time audit and feedback about when allergic-type reactions occur to support efforts to reduce inappropriate and unnecessary antibiotic exposures that increase patient-level harm without improving outcomes.


Reconstruction techniques using autologous hamstring tendons were generally applied for chronic Achilles tendon rupture with a large defect size. Previous studies have reported good clinical results of this technique for young or middle-aged patients, however, the clinical outcomes in older patients have been unclear. This retrospective case series reviewed four male patients aged >70 years  who underwent Achilles tendon reconstruction using the hamstring tendon autograft for chronic rupture of the Achilles tendon with a large tendon defect. The proximal-distal length between the healthy tendon stumps was measured using sagittal T2-weighted magnetic resonance imaging (MRI). The American Orthopedic Foot and Ankle Society (AOFAS) ankle-hindfoot score and postoperative complications were evaluated. The duration from the traumatic event or appearance of symptoms to surgery was 3.8  months. The mean measured gap between the healthy tendon stumps was 67.5 mm on MRI. The AOFAS ankle-hindfoot score improved from 67.3 to 99.5 at the mean follow-up period of 40.3  months. No donor site morbidity was observed in all patients. Re-rupture was detected at the five-month follow-up in one patient who had removed a hinged ankle-foot orthosis with adjustable heel wedges without permission. Achilles tendon reconstruction using a hamstring tendon is a viable option for treating selected patients with chronic rupture of the Achilles tendon with a large tendon defect even in older patients. To improve clinical outcomes, a better understanding should be provided to family members as well as older patients regarding the postoperative rehabilitation program. Achilles tendon rupture is a common tendon injury in the adult population, and its incidence has increased over the past decade . Recent Chronic Achilles tendon rupture is defined as a rupture with delayed diagnosis > 4 weeks from the initial injury . In geneReconstruction techniques using autologous hamstring tendons were generally employed for chronic Achilles rupture with a large defect size . AchilleThis study was approved by the ethics committee of our university. This retrospective analysis included four men aged ≥70 years  who underwent Achilles tendon reconstruction using the hamstring tendon for chronic rupture of the Achilles tendon. All patients were diagnosed with chronic Achilles tendon rupture at primary clinics and were referred to our hospital for surgical treatment. Chief complaints, injury mechanism, cause of chronicity, and the durations from the traumatic event or appearance of symptoms to surgery were reviewed.Surgical treatment was indicated for treating patients with primary chronic Achilles tendon rupture who had a functional impairment, weakness, and palpable gap. Reconstruction of the Achilles tendon using the hamstring tendon was employed for the rupture with a gap length of at least 50 mm on magnetic resonance imaging (MRI). Preoperative MRI was performed in a relaxed supine position with natural plantar flexion of the ankle using a 1.5- or 3.0-Tesla MR system according to each scanning protocol. The proximal-distal length between the healthy tendon stumps was measured in all patients on sagittal T2-weighted MR images. Pre- and post-operative ankle range of motion (ROM), and American Orthopedic Foot and Ankle Society (AOFAS) ankle-hindfoot score was assessed, and postoperative complications were evaluated at the last follow-up as clinical outcomes.Operative techniqueAll surgical procedures were performed by two orthopedic surgeons with >15 years of experience. Each surgery was performed with the patient in the prone position and under general anesthesia. A 3-cm straight incision was created over the pes anserinus, and the semitendinosus or gracilis tendon was harvested using a tendon stripper. A straight longitudinal skin incision was made along the medial border of the Achilles tendon. The proximal and distal tendon stumps were exposed Figure , and thePostoperative managementThe ankle was immobilized in the gravity-assisted equinus position for four weeks under non-weight-bearing conditions using a posterior ankle splint for a week and a below-knee cast for three weeks, respectively. Partial weight-bearing was permitted under hinged ankle-foot orthosis (AFO) with adjustable heel wedges to maintain the plantar flexed position four weeks postoperatively. ROM exercise was started as a physiotherapy program. Patients were allowed limited active ROM from gravity-assisted equinus position to plantar flex position which was defined by the angle of heel wedges. The patients were cautioned to avoid hyperdorsiflexion of the ankle. Subsequently, full weight-bearing was allowed five weeks postoperatively. Initially, four solid heel wedges were fixed on the bottom of the orthosis. One of each heel wedge was removed once a week from seven weeks to 10 weeks. The AFO was utilized to limit hyperdorsiflexion until three to four months postoperatively.Demographic and clinical dataThe demographic and clinical data of all patients are shown in Tables Three of four geriatric patients returned to daily activity at the pre-symptomatic level at four months postoperatively, and their activity level remained unchanged at the final follow-up. Re-rupture was detected at the five-month follow-up visit in another patient.Illustrative case no. 4A 71-year-old man started to feel pain of right posterior calf without particular cause six months earlier and was referred to our clinic. Chronic Achilles tendon rupture was detected, and he underwent reconstruction of Achilles tendon using a semitendinosus tendon graft. Ankle ROM exercise was started after a below-knee cast removal four weeks postoperatively, and partial weight bearing was permitted under AFO. Re-rupture was detected at the five-month follow-up visit, and an inquiry revealed non-use of the AFO postoperatively. As a revision surgery, the rupture site was repaired by suturing, and the Achilles tendon complex was augmented using a knotless suture anchor and suture tape . Although the suture site of the proximal tendon stump and graft was disrupted in this case, the hamstring tendon graft remained normal. Rehabilitation was performed according to the protocol described above. The postoperative course was good without recurrence 27 months after the revision surgery.This study revealed that Achilles tendon reconstruction using hamstring tendons has the potential to improve the clinical status of older patients with large defects and the mean delay in surgery of 3.8 months in chronic Achilles tendon rupture. Nonsurgical treatment options might be reserved for patients with low functional demands or those with significant medical contraindications for surgical treatment . HoweverSurgical reconstruction is appropriate management in most patients with chronic Achilles tendon rupture who have function loss, weakness, and a large gap . SeveralHere, the Achilles tendon ruptures transited to chronicity because the ruptures occurred with minor trauma or non-traumatically, suggesting fragility in the original Achilles tendon of older patients. Less severe degeneration is expected in the harvested graft compared to the local tissue, including the tendon and aponeurosis around the stumps, especially in older patients. Because higher degeneration was identified histologically in the ruptured Achilles tendon than in the healthy tendon . AdditioA superficial incisional surgical site infection was observed in one patient with diabetes mellitus as a minor complication. Re-rupture was detected in another patient at the five-month follow-up visit; however, the postoperative rehabilitation program had not been followed since the below-knee cast was removed. The patient removed the AFO without permission and started walking and performed excessive ankle dorsiflexion. The maximal strain of the Achilles tendon declines with aging, and significantly lower values were noted in participants aged approximately 70 years . Thus, nThe major limitations of this study are the small number of male patients with different rupture sites such as proximal and mid substance, and the lack of a control group. We focused on older patients who accounted for a part of the patient population with chronic Achilles tendon rupture with a large tendon gap. Another technique was employed for chronic Achilles tendon rupture with a small or moderate tendon gap size. Hence, this single-center study did not address large numbers of older patients who underwent Achilles reconstruction using a hamstring tendon. Future prospective, multicenter clinical trials are warranted to investigate the midterm and long-term clinical outcomes of older male and female patients with chronic Achilles tendon rupture. Second, only the AOFAS ankle-hindfoot score, ankle ROM, and postoperative complications were assessed as clinical outcomes. Quantitative functional analysis should be evaluated in future studies, such as isometric muscle strength in plantar flexion and electromyography or tensiomyography in gastrocnemius muscles.Our results suggest that Achilles tendon reconstruction using a hamstring tendon is a viable option for treating chronic rupture of the Achilles tendon with a large tendon defect, even in older patients. Sufficient strength is expected for hamstring tendon autografting compared to the aponeurosis and tendon stumps in the chronic status, especially in older patients. To improve clinical outcomes, better understanding should be provided to family members as well as older patients regarding the postoperative rehabilitation program.


Depression and coronary artery disease (CAD) are often comorbid conditions. The presence of depression significantly interferes with the recovery after CAD therapy intervention, such as percutaneous coronary intervention (PCI), one of the most common medical procedures in developed countries. Brain derived neurotrophic factor (BDNF) has a major role in angiogenesis and neuromodulation. Its levels were previously shown to be reduced in patients with depression, and latest studies indicate similar in patients with CAD. However, the correlation of BDNF levels and depression after CAD treatment is unknown.The aim of this preliminary study is to assess the changes in BDNF levels in patients with depressive symptoms during a six-month period upon PCI.Antidepressant-free participants that underwent PCI with stent placement due to myocardial infarction or angina pectoris were enrolled in the study. Depressive symptoms were evaluated at baseline using the Beck’s Depression Inventory II (BDI-II) with a cut-off score ≥20 indicating moderate depression. Serum BDNF levels were measured from blood samples drawn a day after (baseline) and six-months upon a successful PCI without complications. The t-test for dependent samples was used with marked significant differences at p<0,05.Altogether, 76 participants were included in the study, of which 25 finished a six-month follow-up. Participants with BDI-II≥20 at baseline had higher serum BDNF levels in the second measurement . No significant difference was found in serum BDNF levels in measurements between participants with and without depressive symptoms . Statistically significant difference was found between serum BDNF in the first and second measurement in the overall sample  and in participants with baseline moderate depressive symptoms , but not in those without .Serum BDNF levels in participants with baseline moderate depressive symptoms increased after a six-month period upon successful PCI treatment, whereas that trend was not observed in participants without depressive symptoms. This highlights the potential synergistic role of BDNF in comorbid depression and CAD.None Declared


Plants accomplish multiple functions by the interrelationships between functional traits. Clarifying the complex relationships between plant traits would enable us to better understand how plants employ different strategies to adapt to the environment. Although increasing attention is being paid to plant traits, few studies focused on the adaptation to aridity through the relationship among multiple traits. We established plant trait networks (PTNs) to explore the interdependence of sixteen plant traits across drylands.Our results revealed significant differences in PTNs among different plant life-forms and different levels of aridity. Trait relationships for woody plants were weaker, but were more modularized than for herbs. Woody plants were more connected in economic traits, whereas herbs were more connected in structural traits to reduce damage caused by drought. Furthermore, the correlations between traits were tighter with higher edge density in semi-arid than in arid regions, suggesting that resource sharing and trait coordination are more advantageous under low drought conditions. Importantly, our results demonstrated that stem phosphorus concentration (SPC) was a hub trait correlated with other traits across drylands.The results demonstrate that plants exhibited adaptations to the arid environment by adjusting trait modules through alternative strategies. PTNs provide a new insight into understanding the adaptation strategies of plants to drought stress based on the interdependence among plant functional traits.The online version contains supplementary material available at 10.1186/s12870-023-04273-0. Plant functional traits are measurable attributes that are closely related to plant functions . Plant tPrevious studies revealed the interdependence of multiple traits by employing correlation analyses , 13, strRecent studies applied network analysis to identify both the relationships among plant traits and the hub or key traits of plants under different environmental conditions , 19, 20.Drylands, which are highly vulnerable to climate change, account for approximately 45% of the global land area . They prThe adaptation strategies of plants to drought also depend on their life-forms . ResourcSixteen plant functional traits, of which six economic traits, six chemical traits and four structural traits, were measured in 80 dominant plant species from 83 sites in the dryland ecosystems of China. Network analysis was used to determine: (1) the network relationships of functional traits and their adaptation strategies to drought stress across different arid regions and plant life-forms; (2) the key traits among the sixteen leaf traits; and (3) the connectivity of economic, chemical and structural traits across different arid regions and plant life-forms.This study included 83 field sites across drylands of China Figure . The sitarea), leaf mass per area (LMA), leaf thickness (LT), leaf nitrogen concentration (LNC), leaf carbon concentration (LCC) and leaf phosphorus concentration (LPC)  was obtained with Plant trait networks (PTNs) are biological networks that use plant traits as nodes and relationships between the traits as edges . Firstly to zero . The adjThree network parameters, that is, degree, edge density and modularity, were considered due to their ecological importance . The degThe parameters of PTNs were calculated using the “igraph” package in R. A PTN was established for each bootstrapping by performing 5000 random resamplings to determine the range of uncertainty for these network parameters, and at least three-fourths of all species were selected randomly for each time. In addition, previous studies have shown that the number of species affects the network relationships between traits . Therefoarea, LMA, LT, LCC, LNC and LPC), chemical  and structural  traits, the absolute and relative importance were calculated. The absolute importance was calculated as the average degree of each type of trait, and the relative importance was calculated as the absolute importance divided by the sum of all trait degrees [To compare the importance of economic . A level of P < 0.05 for all, Fig. Based on the dataset of the sixteen plant traits, plant trait networks were constructed and parameters were calculated Fig. a. Edge dP < 0.05) for woody plants (PTNs-woody plants) than for herbaceous plants (PTNs-herbaceous plants), while the modularity of woody plants was greater (P < 0.05) than for herbaceous plants  means that the trait is highly related to other traits in a network, which suggests the trait may regulate pivotal functions affecting the entire phenotype . Stem phTrait centrality was altered within the plant’s life-forms and in different arid regions. Leaf carbon concentration (LCC), an important trait reflecting leaf structural energy costs , 62, wasIn the present study, the sixteen plant functional traits were classified broadly as structural, chemical and economic. Economic traits were more important than the other traits in the plant trait networks across the study area Figs. . NutrienThe importance of economic, chemical and structural traits varied across different life-forms; the connectivity of economic traits was important for woody plants, whereas, the connectivity of structural traits was important for herbaceous plants Fig. . These rThis study provided the first evidence that adaptation strategies of plants in drylands of China are mediated by plant trait networks. Herbaceous plants had a greater edge density and a lesser modularity than woody plants, thus, woody plants are more tolerant of aridity stress than herbs. Herbaceous plants prioritize connections between structural traits and woody plants prioritize connections between economic traits. Plants displayed a greater edge density in semi-arid than in arid regions, but the degree of modularity in arid and semi-arid regions were similar, indicating plants in semi-arid regions could be more efficient for multiple functions than plants in arid regions. Stem phosphorus concentration (SPC) was a hub trait as it shared high connections with all other traits. Changes in trait modules indicated that plants adapted to the local conditions through alternative strategies. In conclusion, by using the plant trait networks (PTNs), this study provided an effective trait-based approach to explore how plants respond to the arid environment.Below is the link to the electronic supplementary material.Supplementary Material 1


Pityriasis rubra pilaris (PRP) is an uncommon, chronic papulosquamous disorder with an unestablished etiology.PRP is often recalcitrant to treatment and there are no US Food and Drug Administration (FDA)-approved treatments. Typical therapies for mild PRP include emollients, keratolytic agents, topical corticosteroids, and topical calcineurin inhibitors. Systemic therapy including oral retinoids and methotrexate have also shown promise for managing symptoms and reducing inflammation in more severe cases. Biologics commonly used in the management of psoriasis, including tumor necrosis factor alpha -inhibitors, secukinumab, and ustekinumab have recently been used for treatment of PRP.Herein, we present a case of pPRP in a patient with adenosquamous carcinoma of the lung.A 72-year-old female with a history of hypertension and prior tobacco use initially presented to the dermatology clinic with a 1-month history of a new rash that began on her hands and spread proximally , Fig 3. Her skin biopsy revealed psoriasiform epidermal hyperplasia with overlying hyperkeratosis and parakeratosis alternating with orthokeratosis both horizontally and vertically. The epidermis displayed spongiosis with a superficial vascular ectasia, while the superficial dermis revealed a sparse lymphocytic inflammatory infiltrate consistent with PRP .Fig 4HisHer screening labs showed a positive QuantiFERON gold and positive Hepatitis C antibody. Due to rapid progression of the rash and severe ectropion from inflammation and edema of the face, she was admitted to the hospital for additional workup and wet wraps. A chest x-ray performed to rule out active TB found a nodule in the right upper lobe of her right lung concerning for a malignancy or infection. She was unable to produce sputum for an acid-fast bacilli stain. Ultimately, a biopsy of the nodule revealed T3N2M0 Stage 3 adenosquamous carcinoma of the lung.In constellation with her skin findings, she was determined to have pPRP in the setting of newly diagnosed pulmonary adenosquamous carcinoma. Treatment options were limited due to latent TB, untreated chronic hepatitis C, and comorbid severe hypertension. The patient was started on clobetasol ointment used twice daily, home wet wraps of topical triamcinolone ointment, hydrocortisone ointment, and bland emollients in addition to acitretin 25 mg daily. Due to cost and perceived lack of benefit, the patient self-discontinued acitretin after 1 week. She did not receive oral prednisone. Despite persistent use of topical steroids, her rash continued to progress for several weeks. She ultimately underwent resection of her adenosquamous carcinoma of the right lung and treatment with wet wraps while hospitalized which resulted in remarkable rapid resolution or her rash.Cutaneous paraneoplastic conditions are a diverse group of disorders that are related to an underlying internal malignancy.Including our case, there are 17 total cases of pPRP in the literature since 1983pPRP is a rare form of PRP associated with solid or hematolymphoid malignancies, which is often recalcitrate to standard treatments. Vitamin A analogues, oral steroids, and topical steroids treat symptoms, but the resolution of the rash usually requires treating underlying the malignancy. Evidence-based data on treatment are limited and largely drawn from case reports.Healthcare providers should consider cancer screening among patients with rapid-onset or treatment-resistant PRP. There are no current guidelines for malignancy workup recommendations in patients with PRP. Clinicians should start with age-appropriate cancer screening, a thorough review of systems, and evaluation of risk factors like a family history of cancer or use of tobacco or alcohol.None disclosed.


Expressional profiling of the endometrium enables the personalised timing of the window of implantation (WOI). This study presents and evaluates a novel analytical pipeline based on a TAC-seq (Targeted Allele Counting by sequencing) method for endometrial dating. The expressional profiles were clustered, and differential expression analysis was performed on the model development group, using 63 endometrial biopsies spanning over proliferative , early-secretory , mid-secretory  and late-secretory  endometrial phases of the natural cycle. A quantitative predictor model was trained on the development group and validated on sequenced samples from healthy women, consisting of 52 paired samples taken from ESE and MSE phases and five LSE phase samples from 31 individuals. Finally, the developed test was applied to 44 MSE phase samples from a study group of patients diagnosed with recurrent implantation failure (RIF). In validation samples (n = 57), we detected displaced WOI in 1.8% of the samples from fertile women. In the RIF study group, we detected a significantly higher proportion of the samples with shifted WOI than in the validation set of samples from fertile women, 15.9% and 1.8% (p = 0.012), respectively. The developed model was evaluated with an average cross-validation accuracy of 98.8% and an accuracy of 98.2% in the validation group. The developed beREADY screening model enables sensitive and dynamic detection of selected transcriptome biomarkers, providing a quantitative and accurate prediction of endometrial receptivity status. Though IVF success rates have improved significantly, many patients still fail to conceive and experience recurrent implantation failure (RIF). The reasons for embryo implantation failure are highly heterogeneous, attributable to the quality of the embryo, the endometrium, and the interaction between the two2. As these circumstances vary from patient to patient, applying a personalised approach for assessing embryo quality and endometrial receptivity could potentially increase the chance of implantation during IVF.Infertility affects millions of people of reproductive age worldwide, and increasingly more couples undergo in vitro fertilisation (IVF) to achieve pregnancy6. In addition, based on transcriptional studies of endometrial biopsies, the WOI can be shifted or displaced over time8. It has been estimated that displaced WOI occurs in around 10% of women undergoing IVF and at least 25% with RIF11. In this light, the personalised determination of the WOI timing could be a standard procedure for women undergoing IVF. This would be crucial to avoid implantation failure after embryo transfer, possibly due to the asynchrony between the endometrial and embryonal development. Therefore, testing the endometrial receptivity and establishing the time for WOI may be particularly beneficial for RIF patients.The period when the endometrium becomes receptive to embryo implantation is called the window of implantation (WOI). There is no consensus yet on the exact timing and length of the WOI period. Generally, the endometrium is considered to become receptive seven days after the luteinising hormone (LH) peak during the natural cycle (NC) and lasts for two days. However, several studies suggest that the length of the WOI may vary from two to up to 6 days12. Besides the altered gene expression profile of endometrial tissue, PCOS patients are often affected by obesity, hyperinsulinemia, and increased general inflammation, likely contributing to infertility13. Nevertheless, to our knowledge, no systematic studies have been published describing the gene expression signature of essential endometrial receptivity genes in PCOS throughout the entire menstrual cycle.Polycystic ovary syndrome (PCOS) is an infertility-associated disorder common among women of reproductive age. It is still unclear whether endometrial receptivity is affected in PCOS patients. The dysregulation of several endometrial receptivity-associated genes in PCOS patients has been previously described15. The hormone-induced regulatory cascade leads to endometrial maturation and major gene expression changes, culminating with the opening of the WOI. The transcriptomic landscape of endometrial maturation is well characterised through whole transcriptome studies where different gene expression profiles have been detected between proliferative (PE), early- (ESE), mid- (MSE), and late-secretory (LSE) endometrium17. Based on these studies, biomarkers for WOI determination and personalised embryo transfer (pET) have been provided. Diagnostic tests based on gene expression profiling of varying sets of endometrial receptivity biomarkers have been developed and integrated into infertility treatment21. Endometrial receptivity tests currently in clinical use are the ERA® test (Igenomix)18, the ER Map® test (IGLS)20, the WIN-Test (INSERM)21, and the rsERT test (Yikon Genomics Company)10. Compared to the targeted sequencing approach used in the present study, these tests are limited either by the sensitivity and dynamic range in detecting transcript abundances or by the scalability of the technique applied.Considerable effort has been made to describe molecular changes in the WOI22, 11 additional genes relevant to WOI, and four housekeeper genes. The computational model was developed using Illumina sequencing-based TAC-seq technology (Targeted Allele Counting by sequencing), enabling biomolecule analysis down to a single-molecule level23, as shown in Fig. This study introduces the beREADY model for reliable WOI detection. The 72 genes analysed with this test contain 57 endometrial receptivity-associated biomarkersFirst, gene expression signatures of 68 endometrial receptivity genes were analysed in the model training and development (MD) group samples (n = 63), consisting of both healthy volunteers and PCOS patients. Based on collected data, a continuous and quantitative three-stage (from pre-receptive to receptive to post-receptive) computational classification model was developed. Next, the model classification accuracy was validated on the model validation (MV) group samples  of healthy women. Finally, the RIF group samples  at MSE) were examined with the validated computational classification model. The outline of this study is presented in the Supplementary Fig. This analysis of endometrial receptivity biomarkers revealed no difference between healthy women and PCOS patients Fig. . ComparaBefore test development, 11 samples from the MD group were excluded from downstream analysis due to inconsistency between histology results and LH-day measurements. The remaining 63 MD group samples clustered clearly according to the menstrual cycle phases and enabled determining the receptivity class prediction Fig. . Based oEndometrial samples with two similar receptivity classes were classified as belonging to a transitionary class. Samples between the pre-receptive and receptive groups were defined as early-receptive and samples between receptive and post-receptive as late-receptive. However, samples positioned in the early-receptive, receptive, or late-receptive groups were collectively considered to represent the normal variability of WOI.Endometrial tissue samples from healthy volunteers (MV group) were used for classification accuracy validation. In total, tissue samples from NC ESE n = 26), MSE (n = 26), and LSE n = 5) phases were analysed Table . The tes6, MSE  were analysed. Out of the RIF MSE samples, three patients  had a pre-receptive profile and displaced WOI Table . No late24, our prediction model allows the construction of the continuous transcriptomic states of endometrial receptivity development.This study presents a novel tool for measuring endometrial receptivity. It is based on a highly quantitative TAC-seq assay that allows precise and cost-effective endometrial receptivity biomarker analysis. A custom computational model for classifying sequenced samples was developed to analyse data generated by the TAC-seq pipeline. As it is based on the expressional profile of a targeted set of genes, the beREADY classification model can be used in high-coverage whole-transcriptome studies. As a result, using endometrial biomarkers discovered from datasets with high predictive power for receptivity25. Therefore, the focus on helping RIF patients has shifted towards elucidating the maternal factors contributing to unsuccessful IVF cycles. This interest has propagated the development of transcriptomic analysis tools for determining the receptivity status of the endometrium21. Those tests use varying sets of targeted genes and different methodologies. The ERA test analyses 238 receptivity genes using microarray technology, Map®/ER Grade® test, and ERPeakSM test analyses 40 genes and WIN-Test 11 genes with quantitative PCR. The selection of genes in the beREADY model is based on the comprehensive meta-analysis of endometrial receptivity biomarkers22, complemented by eleven additional genes relevant for WOI and four housekeeper genes. To our knowledge, the beREADY model is the only endometrial receptivity testing tool that applies the Unique Molecular Identifiers (UMI) technology, enabling original transcript counts estimation while avoiding the PCR-caused bias in results. Therefore, TAC-seq technology eliminates laboratory-caused PCR duplicates and counts the biomarkers at a single-molecule level23. This approach has already been used to determine the menstrual cycle phases from endometrial tissue26.Previous transcriptomic studies of endometrium have shed light on the complex cross-talk mechanism between implanting embryos and the uterus. This insight has explained why embryo implantation in some women repeatedly fails, regardless of the use of seemingly high-quality embryos13, there are no large-scale and systematic studies simultaneously analysing numerous endometrial receptivity-related genes in different cycle phases in PCOS patients. In addition, the endometrial tissues of PCOS patients have demonstrated altered responses to steroids28, but the overall effect of endometrial dysfunction on pregnancy outcomes is still unclear29. Therefore, we tested if PCOS alters the transcriptomic profiles of our selection of receptivity biomarkers. We found no significant differences between healthy controls and PCOS patients in the expression profile of endometrial biomarkers throughout the menstrual cycle from PE to the LSE phase. Based on these findings, we concluded that the selected receptivity biomarkers are unaffected in women with PCOS, and the samples were suitable for use as a reference together with samples from healthy women in the development of the beREADY model. One of the contributing factors to why we did not see significant differences in the expressional profiles of PCOS and healthy samples is that some women in the test development group had lost their PCOS characteristics since their initial diagnosis due to ageing. Nevertheless, it must be noted that PCOS is a lifelong condition despite the effect of ageing and that a retrospective diagnosis is allowed30. Secondly, patients in our study were not obese. When combined, PCOS and obesity can still introduce an additional risk factor for impaired endometrial receptivity31. For these reasons, our conclusions regarding receptivity in PCOS patients are valid for women with PCOS patients within the normal range of BMI.For beREADY model development, we used natural cycle endometrial samples from healthy women and women with PCOS diagnosis. Although the involvement of endometrial factors in PCOS-associated infertility has been suggested 32. In the MSE group, we also classified six samples as early-receptive , suggesting a slight shift in WOI that remained within the normal range of receptivity. Therefore, transcriptomic profiling can offer additional accuracy for determining the individual receptivity timing of the endometrium.We validated the beREADY model by analysing endometrial biopsies from healthy women representing the fertile female population, displaying a high similarity between sample collection time and the molecular test result . This similarity can be explained by the fact that the healthy women in our validation group were all LH tested and had ultrasound confirmation of ovulation and normal hormonal values. Despite this, one MSE sample  with corresponding tissue morphology suggested the PE phase had shifted WOI according to the beREADY. In that case, the beREADY model classified the sample as pre-receptive, suggesting that the WOI had not yet arrived. However, displaced WOI among oocyte donors and women undergoing IVF without RIF diagnosis has been previously reported33. Due to its heterogeneous nature, the causes of RIF have remained largely unknown. When focusing solely on endometrial factors, it has been proposed by Sebastian-Leon et al. that RIF is caused by at least two distinct molecular phenomena of displaced (asynchronous) or disrupted  WOI8. In our study, we applied the beREADY model to detect the rate of displaced WOI in a study group of 44 RIF patients. In total, we detected shifted WOI in 15.9% of RIF cases, which is slightly less than the results described previously by Lessey et al., 25%6, Mahajan et al., 27.5%34, Hashimoto et al., 24%9, and Patel et al. 17.7%25 in women with RIF diagnosis. Compared with the MV group, the RIF study group demonstrated a significantly higher proportion of shifted WOI cases (p < 0.05), indicating that displaced WOI can cause RIF. Moreover, Eisman et al. described a significant decrease in ongoing pregnancy rates in the RIF cohort with asynchronous endometrium compared to control infertile patients with detected WOI displacement35, suggesting that there are additional factors in implantation failure beyond an adjustment in progesterone exposure. Indeed, in RIF cases, additional endometrial-related factors likely contribute to the implantation failure along the displaced or disrupted WOI, like dysbiotic vaginal or uterine microbiome37.The criteria for RIF diagnosis are controversial and include several factors, such as the number of failed treatment cycles, the number of transferred good-quality embryos and maternal age38, Win-test3 and rsERT10 tests. It has also been proposed that receptivity testing before the first embryo transfer increases the implantation and cumulative pregnancy rates when pET is compared with conventional embryo transfers19. Contrary to those reports, recent randomised controlled trials (RCT) and meta-analyses question the effectiveness of endometrial testing and find its impact insignificant42. Although other similar systematic meta-analyses and single cohort studies confirm these results of no benefit of WOI testing in good prognosis patients, they also report that in the RIF subgroup, there is a significant increase in pregnancy rates following pET44. Another study by Haouzi et al. demonstrated that the implantation rate increased over three times, from 7 to 23%, after using the personalised WOI determination in IVF patients with RIF diagnosis3. Despite generally supporting endometrial testing in RIF cases, these studies also stress low certainty of evidence, thus advocating continued research on this controversial topic44. One reason for the disparity between studies might be that the actual rate of recurrent implantation failure due to endometrial factors may be lower than previously thought. A large-scale retrospective study involving nearly 4500 patients undergoing IVF with preimplantation genetic testing for aneuploidy (PGT-A) discovered that only around 5% of the infertile patients failed to achieve clinical pregnancy following three consecutive frozen euploid single embryo transfers45. Embryonic and maternal factors such as embryonal chromosomal aberrations, impaired endometrial receptivity, maternal immune dysfunction, and infertility-associated diseases, like endometriosis and male factors, can all affect the chance of embryo implantation. Therefore, due to the challenges related to the high heterogeneity of the study designs and groups, only specific subgroups of women might benefit from the endometrial receptivity testing. In summary, personalised endometrial dating likely increases the chance of implantation in patients with recurrent IVF failure. At the same time, it is considered an unnecessary ‘add-on’ for patients planning their first IVF treatment. However, the overall efficacy of the approach is still largely unclear due to the disparity between the large-scale studies and the transcriptomic tests applied.Evidence on the clinical effectiveness of endometrial receptivity testing on the IVF pregnancy rate is still controversial. Several studies have demonstrated a significant increase in implantation rate with pET with ERA46. In this study, the authors demonstrated the reproducibility of the endometrial receptivity testing, as seven women underwent ERA testing twice. The second endometrial biopsy was obtained from these women on the same day of their menstrual cycle between 29 and 40 months after the first study cycle. The intercycle variation analysis showed total consistency in the ERA test diagnoses between the same patient's first and second tests. Therefore, it is believed that the gene expression profile of the endometrial receptivity remains consistent in time, at least for the subsequent couple of months after biopsy, allowing the embryo transfer time to be adjusted according to the endometrial receptivity test recommendations. This personalised embryo transfer would allow to select the time period with the peak of endometrial receptivity for embryo transfer. However, as the initial study involved only seven patients, the question about the endometrial profile’s cycle-to-cycle consistency requires further studies.The potential clinical utility of endometrial receptivity testing is also fully dependent upon the consistency of the gene expression profile in subsequent cycles following the endometrial biopsy. Indeed, this claim has been approved by the seminal paper of Díaz-Gimeno et al.48. Recently, we provided proof for RNA biomarker-based minimally invasive endometrial receptivity testing using uterine fluid-derived extracellular vesicles and applying the beREADY endometrial testing model to successfully determine the receptivity status of the samples49. A similar approach would open new possibilities for non-invasive endometrial receptivity testing, which may eliminate the main shortcomings of the endometrial biopsy-based approaches in personalised embryo transfer.Moreover, using the endometrial tissue biopsy for its receptivity testing would exclude the possibility of embryo transfer in the same cycle, thereby prolonging IVF treatment. Considering the possible cycle-to-cycle variation of the window of implantation timing, the endometrial receptivity testing could benefit if it could be analysed non-invasively at the cycle when embryos are planned to be transferred. Receptivity assessment from uterine fluid would circumvent the abovementioned problems, as uterine fluid aspiration can be done before embryo transfer, which is not detrimental to the implantation rate45. Therefore, in our study, the RIF samples with possibly embryo-associated implantation failure were not excluded, likely explaining why in our findings the receptive endometrium was found in around 85% of RIF cases.Some limitations of this study should also be noted. Although the MD set consisted of samples with clearly different expressional profiles corresponding to the different menstrual cycle phases, this study suffers from a relatively small sample size with uneven distribution of samples between the receptivity classes. Furthermore, to estimate the clinical usefulness of the model, an extensive RCT must be carried out among the RIF patients, comparing the implantation, pregnancy, and live birth rates between pET and conventional embryo transfer cycles. Ideally, the RCT should be carried out with euploid embryos following PGT-A, which could rule out the genetic defects of IVF embryos as one of the possible causes of implantation failure. PGT-A has been shown to improve the cumulative IVF pregnancy outcome, avoiding the RIF diagnosis in some patients because of the transfer of the euploid embryos onlyIn conclusion, our results demonstrate that TAC-seq can be successfully applied for transcriptomic endometrial dating and establishing of the WOI. This assay detected distinct profiles of endometrial samples at pre-receptive, receptive, and post-receptive stages. Additionally, we found no significant difference in the expression levels of receptivity biomarkers in healthy fertile women and women diagnosed with PCOS. Based on these findings, we developed the beREADY endometrial receptivity testing tool with a custom classification model for distinguishing between the transcriptional profiles. In our study, we detected an increased rate of displaced WOI cases in the RIF study group compared to the validation group. Consequently, these findings suggest that applying personalised receptivity testing before embryo transfer could potentially reduce the chance of implantation failure in RIF patients.All methods were carried out in accordance with relevant guidelines and regulations' or the 'Declaration of Helsinki'.® digital ovulation test or BabyTime hLH urine cassette (Pharmanova). Endometrial histological evaluation of biopsies was done according to Noyes’ criteria. None of the women had been using hormonal medications for at least three months before the biopsy. All endometrial tissue biopsies were collected using a Pipelle® flexible suction catheter .Three different study groups were used for the training and testing of the beREADY model. The general characteristics of these groups are presented in Table Endometrial samples in the MD group were collected from different menstrual cycle phases of the NC from healthy fertile volunteers (n = 35) and women with PCOS (n = 39) at the Oulu University Hospital (Finland), Table The MV group consisted of 26 pairs of endometrial tissue samples from healthy volunteers at ESE and MSE phases. In addition, we included endometrial tissue samples collected from five healthy volunteers at LSE phase. Therefore, we sequenced 57 endometrial tissue samples from 31 volunteers . DNase I treatment was performed on column using RNase-Free DNase Set (Qiagen). Purified RNA integrity number (RIN) and quantity were assigned with Bioanalyzer 2100 RNA Nano 6000 kit (Agilent Technologies) and Qubit RNA IQ Assay (Thermo Fisher Scientific). Samples with RIN ≥ 7 were considered eligible for further analysis.22 together with four housekeeping genes 23 and 11 additional WOI-related genes .In total, 57 previously published biomarkers were used as endometrial receptivity biomarker genes23 was applied for endometrial receptivity biomarker profiling  was first denatured 2 min at 80 °C and then mixed with 1 µl FIREScript® RT cDNA synthesis MIX . One microliter of TAC-seq probe mixture was added to previously prepared cDNA. After an hour of stringent hybridisation at 60 °C, the thermostable ligase was mixed according to the protocol. Further, PCR was performed in 40 µl volume containing 1 × proofreading HOT FIREPol Blend Master Mix (Solis BioDyne) and 250 nM primers. PCR products were pooled, purified with DNA Clean & Concentrator-5 column kit (Zymo Research), and eluted with 50 µl of elution buffer (EB). Mag-Bind Total Pure NGS beads (Omega Bio-Tek) were added to 50 µl of the purified PCR product, incubated for 5 min at room temperature, and captured by a magnet for 3 min. After incubating, the supernatant was discarded, and beads were eluted in 25 µl of EB. The 180 bp uniform libraries were visualised and quantified on a TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies). High-coverage TAC-seq libraries were sequenced with Illumina NextSeq 550 high output 75 cycles kit.The detailed protocol of TAC-seq was published previously23, and open-source software for TAC-seq data processing is available at https://github.com/cchtEE/TAC-seq-data-analysis. Firstly, the software pipeline matched sequencing reads to the target sequences in the beREADY assay. Up to five mismatches per target sequence were allowed when matching barcodes. Next, UMI thresholding was applied by merging target sequence counts with the same UMI sequence. For the sequencing assay, a UMI threshold of at least one unique UMI per target sequence was used. Finally, after obtaining the molecule count estimates, the counts were normalised considering the geometric mean of the molecule counts of the housekeeping genes. Each sample’s resulting gene expression levels were used for further downstream analysis.TAC-seq sequencing data processing is described in detailThe MD group included endometrial samples collected in different menstrual cycle phases from healthy fertile volunteers (n = 35) and women with PCOS (n = 39) at the Oulu University Hospital (Finland) Table . For the50. Before statistical testing, shifted logarithm transform was applied to the quantified and normalised read counts. The significance of the difference in proportions of displaced WOI in MV and RIF groups was tested with a lower-tailed Fisher’s exact test. In the case of comparative analysis between two groups, an independent t-test with Bonferroni correction was applied to find significant (p < 0.05) differentially expressed genes. A two-sided analysis of variance (ANOVA) was performed when investigating the interaction of two independent variables, and the interaction score was evaluated with the F-test.Statistical analysis, model development and visualisation were done in R language (v4.1.2)For assessing the accuracy of the computational model, fivefold cross-validation was applied. In every iteration, samples were randomly divided into five subgroups, of which four were used to train the model and one to test. At least one random sample from each group was assigned to the testing group during the stratification. The test samples were classified with the most probable receptivity class by comparing every class’s relative receptivity probability outputs. This procedure was repeated 100 times, and the average accuracy over all the classes was reported.51. The development set eigenvectors and scaling parameters from the PCA were used to project MV or RIF samples to the previously selected principal components. After projection, the centroids for the MD receptivity phases were calculated. Subsequently, squared Mahalanobis distances of the projected samples to the reference set group centroids were calculated. Samples with p-values corresponding to the lower tail of the χ2 distribution smaller than 0.025 were considered outliers52. This procedure was repeated for the closest pair of groups. The relative probability of the test sample belonging to either of the closest groups was reported. Only distances to the closest temporally adjacent receptivity stages were compared on the second iteration, constructing a hierarchical sequence of exclusive predictive classes. The relative receptivity class  probabilities for each studied sample were reported.The model for detecting endometrial receptivity was based on relative cluster distances to the MD phase groups after dimensionality reduction with principal component analysis (PCA). Briefly, the MD samples were normalised with the geometric mean of housekeeper gene expression levels and scaled. Next, Horn’s parallel analysis  determined the number of principal components to keepThe study was approved by the Research Ethics Committee of the University of Tartu, Estonia (333/T-6) and by the Research Ethics Committee of the Hospital District of Northern Ostrobothnia, Finland (No 65/2017).Written informed consent was obtained from all individual participants included in the study.Supplementary Information.


BackgroundStaphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) are a recurrent challenge for the Royal Marines at the Commando Training Centre (CTCRM). The intensity of commando training, its impact on skin integrity, and persistent colonisation reservoirs within the training centre have thwarted attempts to prevent these outbreaks.Outbreaks of Panton-Valentine Leucocidin (PVL)-producing methicillin-resistant AimTo present an outbreak of PVL-producing MRSA SSTIs at a military training centre, demonstrating the benefit of additional abrasion and laceration swabs on the identification of colonised personnel and showing the effectiveness of a 10-day decolonisation regime.MethodFollowing the identification of the outbreak of PVL-producing MRSA, all 36 members of the Recruit Troop underwent nasal MRSA screening to identify MRSA carriers. The screening was repeated on day 16 after completing an enhanced 10-day decolonisation regime. A third screening was conducted on the 110th day after a second peak of infection was identified. Various infection control measures, such as enhanced cleaning, restriction of movement and adjustments to the military training serials, were introduced to prevent further spread through the training centre.ResultsIn this outbreak, two-thirds (eighteen) of the Recruit Troop suffered MRSA-PVL skin infections requiring antibiotic therapy and three required hospital admission for surgical management of their abscesses. The outbreak lasted 130 days, with two spikes in infections 10 weeks apart. The outbreak was successfully confined to one troop.ConclusionWith concerns about low identification rates of carriers using nasal screening for MRSA, in this outbreak, we improved the identification of asymptomatic carriage with the simple step of additional culture swabs for all cuts and abrasions. Improved identification of colonised recruits, along with an enhanced decolonisation regime and rigid infection control practices, prevented the further spread of the clone through the training centre.In a population with constant ongoing skin trauma, such as the military, contact sport athletes and iIV drug users, our results show that a culture of suitable abrasions/lacerations will improve the identification of MRSA colonisation compared with nasal swabs alone. Despite ongoing skin trauma and the logistical difficulties in delivering effective decolonisation during military training, decolonisation was successful in 79% of recruits after one decolonisation and 87% after the second 10-day decolonisation.  Staphylococcus aureus (MSSA), there have been regular outbreaks of Panton-Valentine leukocidin (PVL)-producing MSSA and methicillin-resistant S. aureus (MRSA) SSTI [Skin and soft tissue infections (SSTIs) are a common and debilitating problem for the recruits at the Commando Training Centre Royal Marines (CTCRM). Whilst the majority of SSTIs are due to meticillin-sensitive SA) SSTI . ResistaSA) SSTI .Additionally, colonised recruits at CTCRM are at high risk of spreading MRSA amongst their troop  recruits, 30-60 in size with a shared living area) due to regular skin-to-skin contact with colleagues, crowded living conditions and informal sharing of items such as towels. A challenging consequence of commando training, and undoubtedly one of the most significant risk factors for skin infections at CTCRM, is the constant presence of cuts and abrasions on the skin of recruits (‘Woodbury rash’) [S. aureus is carried by approximately one-third of the population, with around 20% being continually colonised, while other individuals are only intermittently colonised [olonised -6. The colonised ,8, and tolonised ,6,8-10.On average, 1300 recruits and 400 potential officers attend training courses at CTCRM annually. In this paper, we present the management of an outbreak of PVL-producing MRSA within an RM Recruit troop and the impact of swabbing broken skin in addition to the traditional nose and throat screening.Two-thirds of the RM recruits in the troop suffered PVL-MRSA skin infections requiring antibiotic therapy. Each infected recruit missed an average of 17 days of training. Three of the troops required hospital admission for surgical management of their abscesses. The outbreak lasted 130 days over the autumn and winter of 2018/19, with two spikes in infection 10 weeks apart.Identification of outbreakWound swabs, routine for all clinical presentations of SSTI, identified two recruits within a single troop with MRSA infection. Both isolates were resistant to gentamicin, ciprofloxacin, clindamycin, erythromycin, and trimethoprim and sensitive to rifampicin, mupirocin, linezolid and doxycycline. Isolates referred to the National Staphylococcal reference laboratory at Colindale, London, were confirmed to be Sequence Type (ST) ST22 CC 22, t309 and carried the PVL gene. ST 22 has recently been described as causing an outbreak within a surgical ward in a UK hospital setting . There aControl measuresAn outbreak team from the Training Centre coordinated the introduction of infection control measures to prevent further spread. Items such as sleeping bags with a high fomite burden but damaged by high-temperature washes were exchanged for new ones.The troop remained housed in their multi-person dormitory when not on outdoor training, occupying the entire floor of an accommodation building. They did not share toilets or showers with any recruits from different troops or classes. At the declaration of an outbreak, the entire floor underwent a deep clean by an external cleaning company, wiping down all surfaces with chlorine-releasing disinfectant to remove dust and inactivate organisms, along with changing and disposing of laundry, bedding and curtains. An enhanced daily environmental cleaning protocol, including disinfectant product cleaning of all high-touch surfaces, was undertaken by the troops and supervised by appropriately trained military staff.The recruits were allowed increased access to the camp's launderette, which was given clear instructions on washing temperatures and handling of bedding and clothing. Enhanced hand hygiene measures were introduced, with monitored hand washing and surface cleaning at the shared canteen and gymnasium.Finally, since the normal commando training program involves repeated periods of exercising in remote training areas for days on end, their program was adapted to allow regular access to shower facilities to facilitate the triple therapy decolonisation.Screening recruits for MRSA colonisationAmies medium with charcoal nose and throat swabs were performed as a self-swab, supervised by nursing staff or Royal Navy/Royal Marine Medical Assistants on Days 1 and 16 (after first decolonisation) for all recruits. Further swabs of the nose/throat/wound were performed on day 110 after the medical team identified new clinical infections indicating a resurgence of the outbreak. Skin swabs were not performed on recruits with intact skin.MRSA screening cultures were performed using MRSA-selective agar (Biomerieux). Incubated as per standard operating procedures at 370 C, plates were inspected after 24 hours and 48 hours of incubation. Suspicious colonies were then characterised by matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) (Biomerieux), and confirmation of PBP2A was confirmed by immunochromatographic assay (Alere PBP2A SA test). Screening for PVL-associated lukS-PV/lukF-PV genes was performed by real-time multiplex PCR, with molecular characterisation by spa typing undertaken by the national reference laboratory. Treatment of SSTIPersonnel with identified SSTI requiring antibiotics were treated as per the CTCRM Infection Policy with flucloxacillin and clindamycin or had case-specific advice from a Consultant Microbiologist. Repeated point-of-care CRP testing was performed on all patients with deranged clinical observations. Recruits requiring additional care and support, such as surgical drainage and IV antibiotics, were admitted to local secondary care.Decolonisation regimenAll RM recruits undertook our local 10-day 'triple-agent' empirical decolonisation regimen repeated a month later. The 10-day regimen  was evolved in 2018 from the traditional five-day MRSA decolonisation hospital regimen for two reasons. Firstly, an increased failure rate has been noted, particularly with PVL-producing Staphylococcal strains in patients over the years. Secondly, the practical and logistical difficulties of effective decolonisation of troops undergoing training in the field were observed. All recruits received a verbal brief and demonstration from a member of the Nursing team and were given Patient Advice Leaflets on MRSA decolonisation.The outbreak lasted 130 days, with peaks in infection two months apart. Thirteen recruits suffered infections requiring antibiotics during the first peak, with many developing a repeat infection in the second peak. Initial decolonisation success rates were higher than expected; however, the second infection peak indicates that recolonisation quickly developed  had wounds sampled initially, twenty-five per cent (9/35) had wounds when the second set of swabs was taken, and eleven per cent (5/42) had wounds swabbed at the third sampling.This outbreak highlights the practical difficulties in assessing and control of an outbreak since moving in and out of the troop by recruits over the 32 weeks was unavoidable since some recruits failed to progress through fitness and military tests or suffered injuries that required time out in rehabilitation, being "back-trooped". Three recruits that left commando training during the outbreak did not complete the full swab or decolonisation regime. Nine recruits joining the troop midway through the outbreak missed the first set of screening swabs, but none had positive swabs at any stage. Four recruits were transferred to other troops for further military training during the outbreak.With a regular intake of new recruits from the UK and overseas, RM recruits are always at high risk of acquisition of new Spa types of S. aureus. Rapid and accurate identification of colonised recruits is essential to enable decolonisation, prevent further spread, and ensure the correct treatment of any SSTI. Previous studies of RM recruits at CTCRM demonstrated surprisingly low levels of nasal carriage of staphylococci despite high rates of concomitant SSTI .Our results show that a culture of suitable abrasions/lacerations will improve the identification rate of those with MRSA colonisation over nasal swab culture.There has been little change to the recommended wound care since the 1979 outbreak of Streptococcus pyogenes involving 1300 recruits. The outbreak was curtailed by education and encouraging troops to identify lesions, self-administer povidone-iodine spray to wounds and use medicated soap .We believe the high success of MRSA decolonisation observed during our outbreak was due to three key factors, namely a very experienced RM training team's policing of the recruits' skin condition, a 10-day triple decolonisation protocol, and regular intensive patient education. A challenge of decolonisation in active military personnel is ongoing skin trauma during and after decolonisation and lack of showers when training in field conditions overnight. The RMs are investing in new clothing and equipment to reduce the impact of SSTI, which will allow for improved skin healing. Whilst smoking has been associated with a lower rate of staphylococcal colonisation , we saw Limitations of our study include the lack of throat swabbing at the start of the outbreak, which likely contributed to an underestimate of initial MRSA carriage. Staphylococcal throat carriage is more common in our population age group and is reported in 20% of military recruits ,16. HoweLooking aheadScreening for MRSA using conventional culture and nasal swabbing alone overlooks some colonised patients, incidental carriers or hidden reservoirs . We needIn a population with ongoing skin trauma, such as the military or intravenous drug users (IVDU), our results show that a culture of suitable abrasions/lacerations will improve the identification of MRSA colonisation compared with nasal swabs alone. Moreover, it might be useful to further investigate repeated MRSA decolonisation regimens of longer duration.


Quantification of disease involvement could be useful in decision-making for additional extended mesenteric resection. We investigated the feasibility of magnetic resonance elastography (MRE) of the mesentery and if MRE is capable to detect fibrotic disease involvement of mesentery in active CD.Creeping fat is a pathological feature of small bowel Crohn’s disease (CD), with literature suggesting that bowel resection with extended mesenteric resection is related to less postoperative recurrences. Conventional imaging is unable to accurately quantify the disease involvement (U-test). Within CD patients, the affected and “presumably” unaffected mesentery were compared (Wilcoxon-signed rank test). Repeatability was tested in 15 HVs . Spearman rank correlations were used to investigate the relation between microscopically scored amount of mesenteric fibrosis and viscoelastic parameters.Multifrequency MRE yielded spatial stiffness  and fluidity maps (φ). Viscoelastic properties of seven CD patients’ mesentery were compared to age- and sex-matched healthy volunteers (HV) . Strong correlations were found between percentage of area of mesenteric fibrosis and SWS and |G*| (p < 0.010). No differences were found within CD between affected and presumably unaffected mesentery. Repeatability of SWS showed 95% limits of agreement of  and within-subject CoV of 5.3%.SWS, |G*|, and MRE may have the potential to measure fibrotic disease involvement of the mesentery in CD, possibly guiding clinical decision-making with respect to extended mesenteric resection.NL9105, registered 7 December 2020.Dutch trial register, MRE may have the potential to measure the amount of mesenteric fibrosis of the affected mesenteric fat in active Crohn’s disease, giving more insight into disease progression and could potentially play a role in clinical decision-making for extended mesenteric resection.• MRE of the mesentery in patients with active CD is feasible.• Fluidity and stiffness of the mesentery increase in active CD, while stiffness correlates with the histopathological amount of mesenteric fibrosis.• MRE provides biomarkers to quantify mesenteric disease activity in active CD.The online version contains supplementary material available at 10.1186/s41747-023-00366-5. Creeping fat is a pathological feature of small bowel Crohn’s disease (CD) at cross-sectional imaging and histopathology. It constitutes thickened, hypertrophic mesenteric adipose tissue (hereafter: mesentery) that wraps around the affected bowel wall. The exact mechanism of creeping fat is still debated and has been hypothesized as follows: 1) a passive and protective response to a dysfunctional gut barrier  a passiv.i.e., fibrosis) could facilitate this decision-making.The presence of creeping fat at imaging is not an innocuous finding as it is associated with fibrosis and stricture formation of the affected bowel –6. Currei.e., fibrosis) of the mesentery.Several studies have tried to quantify creeping fat either directly , 13 or ii.e., fibrosis) and adipocyte hyperplasia , , and , respectively. Within-subject CoV were 8.7%, 8.4%, and 14.0%, respectively. Bland–Altman analysis of the repeatability in healthy volunteers is shown in Fig. In the repeatability population  had penetrating fibrosis at histopathology defined as fibrosis that was continuous through all intestinal layers with extension into the mesenteric fat , 29.i.e., fibrosis).The amount of mesenteric fibrosis at histopathology (mesenteric fibrotic score) showed a strong positive linear relation with stiffness (SWS) and shear stiffness (|G*|) measured by means of MRE within active CD patients. This suggests that measuring the SWS and |G*| with MRE could be used to determine the amount of mesenteric fibrosis in patients with active CD. This also suggests that MRE could potentially be used as a biomarker, which gives insight into the degree of mesenteric disease involvement  in active CD patients. Moreover, the spin-echo echo-planar imaging sequence requires fat suppression to reduce EPI artifacts due to high fat signal. Smaller fat signals in proximity to the main water peak are not suppressible with spectral presaturation with inversion recovery yet cause a minimal water-fat shift. The remaining signal is lower, but is not zero as a result of incomplete fat suppression. Nonetheless, the OSS-SNR in subjects was of sufficient amount to determine the viscoelastic properties and notably larger in affected mesentery in CD patients. This could be due to infiltration of other tissues  in the mesenteric fat. Therefore, differences in viscoelastic properties between active CD patients and healthy volunteers are caused by differences in underlying tissue structures.First, we performed an exploratory study with a small patient cohort. This precludes firm conclusions, although the findings suggest a role of MRE in determining the amount of mesenteric disease involvement .


Lingual foregut duplication cysts are rare developmental anomalies with varied symptoms, and adenocarcinomas may develop from them. Radiological evaluations, including computed tomography and magnetic resonance imaging, can help diagnose these lesions and determine their extent. Abnormal enhancement should raise the suspicion of malignancy of a lingual foregut duplication cyst. Early detection and complete surgical resection of lingual foregut duplication cysts are necessary to prevent malignant transformations. Complete tumor resection with adjuvant radiotherapy or radiochemotherapy is recommended. The teaching point is that mucinous adenocarcinoma can develop from a lingual foregut duplication cyst, and radiological studies may aid in the diagnosis.Adenocarcinomas can develop from lingual foregut duplication cysts, which are rare developmental anomalies, and radiologic studies may aid in the diagnosis. Congenital cystic lesions with foregut-derived epithelial linings, known as foregut duplication cysts, can manifest anywhere from the oral cavity to the anus . LingualA 63-year-old male presented to the outpatient clinic of the otorhinolaryngology department with complaints of the sensation of mouth fullness and dysphagia that had lasted for three months. On physical examination, the tongue showed bulging contour with a normal overlying mucosa.Enhanced neck computed tomography (CT) and magnetic resonance imaging (MRI) revealed a well-circumscribed lobulated cystic mass in the tongue, measuring approximately 6.7 × 5.7 × 5.1 cm. CT images showed a low-attenuation mass with multifocal internal calcifications, and septa-like structures were observed within the mass . On neckThe initial radiologic diagnosis of this mass was a lingual foregut duplication cyst, with differential diagnoses of lymphatic malformation and dermoid cyst. Given the impression of a benign mass, the surgeon proceeded with trans-oral resection surgery. After histopathological analysis of the excised mass, the final diagnosis was mucinous adenocarcinoma , and tumThe patient underwent positron emission tomography/CT scan to exclude the possibility of a metastatic lesion from a primary intestinal malignancy, and there was no evidence of a metabolically active lesion in any location other than the resection margin of the tongue . Upper eAfter a multidisciplinary discussion, total glossectomy was planned as a treatment plan, and adjuvant radiochemotherapy was considered a subsequent treatment regimen.Lingual foregut duplication cysts are uncommon anomalies that occur as a result of remnant heterotopic rests of the foregut-derived epithelium after the first trimester of embryological development . Althoug35Radiologic evaluations can help diagnose these lesions and determine their extent. In our case, enhancements of the peripheral wall and internal septations of the lingual foregut duplication cyst were observed on radiologic images, and these imaging findings may reflect the malignant transformation of foregut duplication cyst.Although no standardized treatment protocol has been established for adenocarcinomas arising from foregut duplication cysts because of their rarity, complete resection with adjuvant radiotherapy or radiochemotherapy is recommended .