Prokaryotic systematics is one of the most progressive disciplines that has embraced technological advances over the last century. The availability and affordability of new sequencing technologies and user-friendly software have revolutionised the discovery of novel prokaryotic taxa, including the identification and nomenclature of uncultivable microorganisms. These advances have enabled scientists to resolve the structure of complex heterogenous taxon and to rectify taxonomic status of misclassified strains due to errors associated with the sensitivity and/or reproducibility of phenotypic approaches. Time- and labour-intensive experimental characterisation of strains could be replaced with determining the presence or absence of genes or operons responsible for phenotypic and chemotaxonomic properties, such as the presence of mycolic acids and menaquinones. However, the quality of genomic data must be acceptable and phylogenomic threshold values for interspecies and supraspecies delineation should be carefully considered in combination of genome-based phylogeny for a reliable and robust classification. These technological developments have empowered prokaryotic systematists to reliably identify novel taxa with an understanding of community ecology and their biosynthetic and biodegradation potentials. Prokaryotes are abundant in nature with an estimated 2.2–4.3 million species, of which only ∼21,000 have been characterised and validly published . The dishilaceae  and withacterium . Furtheracterium , suggestTo overcome these limitations, prokaryotic systematists have embraced and integrated whole genome sequence-based analyses in their practices . Not onlThe modernisation of the practices of prokaryotic systematics was not possible without significant contribution and tireless efforts of the scientific community continuously developing bioinformatic tools to help systematists with limited computational experience. The availability of interactive user-friendly bioinformatics tools and resources played an important role in facilitating the smooth integration of whole genome sequences into the discipline. For example, EzBioCloud and Type (Strain) Genome Server (TYGS) are extensively used resources by prokaryotic systematists. EzBioCloud has an integrated sequence database of high-quality 16S rRNA gene and genome sequences of archaea and bacteria . The ser2http://enve-omics.ce.gatech.edu/g-matrix/). OrthoANI is another great tool that calculates ANI values from a set of orthologues genes within the dataset to address the bias introduced by the variation in the size of sequence alignments by pairwise reciprocal similarity criteria [Several taxogenomic approaches are available to assist systematists in identification of novel taxon by comparing genome sequences. One of the most used approaches is the calculation of average nucleotide identity (ANI) between pairs of genomes where genome sequences are spliced into 1020 bp fragments followed by BLAST-search  against criteria . Howevercriteria . The relcriteria ,32,33.http://enve-omics.ce.gatech.edu/g-matrix/) with a user-friendly interface could be used to calculate AAI values for up to 50 genomes. Another efficient and easy to use tool for AAI calculation is EzAAI pipeline, which uses hierarchical clustering tool MMseqs2 [Pairwise average amino acid identity (AAI) values are often treated to be more reliable, particularly for circumscribing higher taxa ,34,35. A MMseqs2  for a se MMseqs2 . The AAIAn important approach for species delineation is the calculation of digital-DDH (dDDH) values using an efficient user-friendly web interface, genome-to-genome distance calculator ,41. The Klebsiella pneumoniae genomes were compared; however, this ratio was decreased to 79% and 72% when Klebsiella pneumoniae subsp. rhinoscleromatis and K. pneumoniae subsp. ozaenae were successively compared [Klebsiella mobilis, Klebsiella variicola and Klebsiella oxytoca genomes were compared with the six Klebsiella pneumoniae genomes, leading to the conclusion that K. pneumoniae subsp. rhinoscleromatis and K. pneumoniae subsp. ozaenae should be treated as distinct species [A pangenomic approach that is based on the calculation of the percentage of the core/pan-genome ratio between different strains is emerging as an important tool for reclassification of bacterial species ,44. Distcompared . A reduc species .−5. The hypothesis is that at least 50% of the genome wide proteome will be shared by two individual species belonging to the same genus [A potential ANI threshold of ∼75% has been suggested for separating different genera . Howeverme genus .Phylogenetic analyses are important to infer evolutionary relatedness among different strains that help systematists in assigning species to correct taxon. PhyloPhlAn 3.0 is a highly efficient approach to rapidly construct phylogenies from large genomic datasets . The pro3Actinomycetota (former known as Actinobacteria), remained unresolved until the integration of genomics [Rhodococcus, a heterogeneous actinobacterial taxon that include species of ecological, industrial and medical importance, encompasses multiple species-groups which equate to the rank of distinct genera [Bacteroidota (formerly known as Bacteroidetes) resulted in elevation of the families Balneolaceae and Saprospiraceae to a new phylum Balneolaeota, and addition of a new class Saprospiria [Flexibacter resulted in defining new monophyletic genera with emended species descriptions [The taxonomic structure of large complex taxon, such as the phylum genomics . The phygenomics . Rhodocot genera ,22. A phrospiria . Similarriptions .Mycobacterium sensu lato was divided into five monophyletic clades designated as “Fortuitum-Vaccae”, “Terrae”, “Triviale”, “Abscessus-Chelonae” and "Tuberculosis-Simiae", which correspond to four novel genera Mycolicibacterium, Mycolicibacter, Mycolicibacillus, Mycobacteroides, respectively [. The "Tuberculosis-Simiae" clade encompasses pathogenic strains including Mycobacterium tuberculosis, which is responsible for tuberculosis in humans. Similarly, Burkholderia cepacian complex containing strains that are responsible for fatal lung infection in humans, was resolved into 36 groups with a reassignment of 22 species to the correct rank and identification of 14 novel species [Phylogenomic analyses also resolved the taxonomic structure of clinically and ecologically important taxa. The genus ectively . The "Tu species .Frankia is known for ecological and agricultural significance but the taxonomic relatedness between Frankia species remained vague [Frankia species were shown to form four distinct groups with capacities to produce plant growth promoting hormones, bioremediation potential, and potential to produce novel bioactive compounds [Streptomyces, Kitasatospora and Streptoacidiphilus, which are well known for their ecological and genetic diversity [Kitasatospora for novel antibiotic discovery [The genus ed vague ,54. Usinompounds . Phylogeiversity . These giversity . In addiiscovery . Therefo4in vitro biochemical assays or API 20/CHL strips provide an overview of the metabolic features of microorganisms but are sometimes sensitive to minor variations in experimental conditions, resulting in poor reproducibility and/or ambiguous data. But do we really need to continue with the traditional phenotyping methods?Traditional phenotyping methods using Turicella which only differed from Corynebacterium strains by lacking mycolic acids and quinone profiles [Turicella otitidis, the sole Turicella species, was reclassified as Corynebacterium otitidis based on phylogenetic analyses [Some studies reported a lack of consistency between chemotaxonomic and phylogenetic data; for example, the genus profiles ,56. Genoprofiles . Turicelanalyses . Therefoanalyses .High throughput phenotyping technologies such as Biolog Phenotype MicroArrays, have been extensively used for characterisation of metabolic pathways in combination with the genomic data, which has also been useful for taxonomic studies . This sy5Candidatus Parcubacteria and Candidatus Microgenomates) [Despite the importance of non-cultivable microbes in maintaining different ecosystems, our knowledge of the microbial world is mostly limited to cultivable taxa. Only ∼2% bacteria from nearly half of the identifiable bacterial domains are culturable . Environnomates) . Signifinomates) . In linenomates) . Assessinomates) . Therefo6The technological advancements and availability of user-friendly phylogenomic tools have clearly benefited prokaryotic systematists. However, there are still some challenges, which need addressing. The most important issue is the quality of the sequencing reads and assembled genomes. More than 2 million entries in the GenBank were found to be contaminated in a recent study, including 4-8% of data for bacterial species . It is iCorynebacterium strains from yellow-eyed penguins belonging to two distinct lineages were proposed to be assigned to two subspecies despite dDDH values being below the 70% threshold due to ANI and AAI values that were higher the 95% cut-off, very close phylogenetic relationship and their association with diphtheritic-stomatitis [While phylogenomic tools provide usable matrices, the threshold values suggested for interspecies and supraspecies delineation should be carefully considered in combination of genome-based phylogeny . For insomatitis .The publicly available resources allow users to analyse smaller number of genomes that are often limited to 50-75 genomes. It causes problems for scientists resolving the taxonomic status of large and complex taxon where these programs need to be installed on Unix/Linux based high-performance computers and data needs to be analysed using the command lines. It requires significant investment in computational resources and advance knowledge to configure and run phylogenomic analyses that may not be feasible for all prokaryotic systematists.7According to the polyphasic approach, groups of phylogenetically closely related strains with similar phenotypic characteristics, ≥70% pairwise DNA-DNA hybridization (DDH) values and >98.7% identity between their 16S rRNA gene sequences are often assigned to the same species . The defPhylogenomically, a prokaryotic genus is defined as a monophyletic group of species sharing at least 50% of genome wide proteome . A group of phylogenetically closely related strains with ≥95% ANI and/or AAI values and dDDH value of ≥70% belongs to the same species. Therefore, genome based phylogenomic metrices could be used for interspecies and supraspecies delineation.i.e., presence or absence of genes involved in phenotypic properties, where possible. It is probably the time to accept the phylogenomic description of novel taxa (species and genera) for validation where quality of genome assemblies is excellent and robust phylogenomic analyses present compelling evidence without extensive polyphasic characterisation (phenotypic and chemotaxonomic properties).In summary, prokaryotic systematics is clearly a progressive discipline that has evolved over a century by embracing advanced technologies. While polyphasic approach provides important information on the cellular composition and lifestyle of an organism, these techniques sometimes show poor reproducibility between different laboratories. Since whole genome sequencing has integrated well into prokaryotic systematics, more emphasis could be placed on describing the genetics, Imen Nouioui: Data curation, Writing – original draft. Vartul Sangal: Conceptualization, Data curation, Writing – original draft.


Venovenous extracorporeal membrane oxygenation (ECMO) is recommended for the treatment of critically ill patients with acute respiratory distress syndrome due to coronavirus disease 2019 (COVID-19). However, ECMO management can cause both bleeding and thrombotic complications. There are insufficient coagulofibrinolytic data for appropriate ECMO management in patients with COVID-19.A 48-year-old man with severe COVID-19-acute respiratory distress syndrome underwent long-term venovenous ECMO management for 48 days. Refractory oronasal bleeding developed on day 13, so the administration of unfractionated heparin was ceased for 29 days.2-plasmin inhibitor complex elevations, suggesting the development of ECMO-induced coagulopathy.The patient showed dynamic coagulofibrinolytic responses associated with ECMO management, as shown by fibrin/fibrinogen degradation products, soluble fibrin, thrombin-antithrombin complex, and plasmin-αWe assessed coagulofibrinolytic markers to decide the appropriate timing for controlling excessive activation of coagulation by exchanging ECMO circuits. Moreover, viscoelastic hemostatic assays were used for adequate transfusion of blood products.Safe long-term ECMO management was completed, which was withdrawn on day 48. The patient was weaned off mechanical ventilation on day 57 and was transferred to another hospital for rehabilitation.Monitoring the coagulofibrinolytic status using markers and viscoelastic hemostatic assays may be effective for safe long-term ECMO management even without anticoagulant therapy. Generally, ECMO management for patients with severe ARDS includes continuous and systemic anticoagulation to prevent thrombosis of the ECMO circuit. However, ECMO management causes both bleeding and thrombosis, and further COVID-19-induced coagulopathy appears to complicate both manifestations. There is insufficient clinical data that include coagulofibrinolytic responses to establish management for hemostasis including anticoagulation in patients with COVID-19 receiving ECMO. Herein, we report the case of a COVID-19-ARDS patient who received long-term VV-ECMO management without anticoagulant therapy because of a refractory bleeding complication. Our case illustrates the dynamic coagulofibrinolytic response that might indicate the transition from hemostasis to systemic coagulopathy. The clinical findings of our case were consistent with the definition of disseminated intravascular coagulation (DIC). Assessment of the coagulofibrinolytic condition using markers and TEG6s® was effective in minimizing thrombotic and bleeding complications.Venovenous extracorporeal membrane oxygenation (VV-ECMO) is recommended for the treatment of severe and critically ill patients with acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 (COVID-19).A 48-year-old man with a height of 171 cm and a body weight of 87 kg presented with fever (38°C). Four days after symptom onset, the patient was admitted to an emergency hospital with progressive dyspnea and diagnosed with COVID-19 pneumonia based on severe acute respiratory syndrome coronavirus 2 polymerase chain reaction. Chest computed tomography (CT) scan revealed bilateral ground-glass opacities and consolidation. His pneumonia worsened under oxygen therapy and the administration of steroids and remdesivir for 8 days; therefore, he was intubated and transferred to our university hospital, the local designated center for patients with COVID-19-ARDS in Ehime, Japan.2/FiO2 ratio 61.9, PaCO2 51.9 mm Hg). Thus we decided to establish VV-ECMO. Cannulation was carried out using a 25 Fr drainage cannula via the right femoral vein and a 19 Fr inflow cannula via the right jugular vein. Initial ECMO settings were sweep gas flow 5.0 L/min, FiO2 100%, blood flow 5.3 L/min, and rotation speed 2050 rpm, and regulated according to his respiratory status. We commenced continuous administration of unfractionated heparin (UFH) titrating to achieve an activated partial thromboplastin clotting time (APTT) ranging from 50 to 60 seconds, although anti-factor Xa monitoring was not available in our hospital. While on ECMO we performed very low-pressure and low-volume ventilation to provide “lung rest,” and also provided prone positioning.On arrival . An ECMO circuit change resolved hemolysis, and apparent thrombus formation was observed within the pump head. Subcutaneous emphysema developed after the circuit change. Contrast-enhanced CT revealed subcutaneous and mediastinal emphysema, but no thrombus formation. After tracheostomy on day 13, refractory oronasal bleeding was caused by contact with the feeding tube and the tracheal tube developed . Although the patient had had negative severe acute respiratory syndrome coronavirus 2 polymerase chain reaction results twice, the bleeding persisted despite cauterization and oronasal packing; therefore we decided to discontinue the administration of UFH on day 20. After that, the coagulofibrinolytic markers increased and sustained elevated levels . On day 24, we performed a planned ECMO circuit change, and minimal areas of peripheral thrombus formation were observed on the oxygenator membrane. Although the increased coagulofibrinolytic activity mildly improved, oronasal bleeding continued despite treatment and transfusion of plasma and PLTs for hemostasis. We evaluated the coagulofibrinolytic status using TEG6s®. It revealed mild hypocoagulability even under transfusion, and fibrinolytic shutdown whereas the markers showed increased fibrinolytic activity  were observed. Considering that it was necessary to correct the increased coagulation activity without resuming UFH for refractory bleeding, we changed the ECMO circuit. The coagulofibrinolytic activation subsequently improved and the FDP value decreased to a normal level. Only minimal areas of peripheral thrombus formation were observed on the oxygenator membrane. The evaluation using TEG6s® before and after the ECMO circuit change confirmed the improvement in coagulability with the transfusion of plasma and PLTs against consumption coagulopathy  and consumption of coagulation factor and PLTs . Improvement in the increased coagulation activity was thought to be required; however, ECMO was withdrawn due to improvement in the respiratory condition, and the patient was successfully decannulated on day 48. Apparent thrombus formation was observed within the pump head and the oxygenator membrane. Coagulofibrinolytic activation was normalized immediately after withdrawal from ECMO, and the oronasal bleeding also improved.The patient was weaned off mechanical ventilation on day 57 and transferred to another hospital for rehabilitation. No obvious thrombotic findings were detected on CT images during the course of the treatment. In such situations, thrombosis is one of the main complications during ECMO management; therefore continuous anticoagulation is recommended. Paradoxically, a high risk of bleeding complications coexists; therefore, adequate anticoagulation management is required. In the previous reports, the median duration of ECMO for COVID-19 was reported to be 13.9 days. Some reports have demonstrated that good clinical outcomes can be achieved even after long-term ECMO treatment. Furthermore, a systematic review of anticoagulant-free ECMO revealed an incidence of circuity and patient thrombosis comparable to those in patients receiving continuous systemic anticoagulation. However, the median duration of ECMO management without any systemic anticoagulation was 4.75 days in this review, which was much shorter than the 29 days in our case.In this study, we found that monitoring coagulofibrinolytic status using markers and viscoelastic hemostatic assays (VHAs) may have been effective for long-term safe ECMO management in a patient with severe COVID-19 ARDS who could not use anticoagulation due to oronasal bleeding. ECMO has been considered a candidate for the effective management of patients who do not respond to optimal conventional mechanical ventilation in COVID-19. Under this situation, abrupt FDP elevations with marked coagulofibrinolytic activations were detected, which might indicate the transition from hemostasis to systemic coagulopathy. When blood is exposed to the non-endothelial surface of the ECMO circuit, hemostatic alterations including coagulation activation and inflammation occur. Hemostatic equilibrium under ECMO is maintained by reserve capacity and administration of an anticoagulant agent. However, the synergistic effect of endothelial dysfunction and inflammation due to causative or concomitant diseases interferes with the balance and causes disequilibrium with resultant bleeding or thrombosis: ECMO-induced coagulopathy (EIC). Furthermore, extensive cross-talk between coagulation and inflammation caused by continuous exposure to ECMO circuit and causative disease can amplify each other and evoke exacervation of EIC. Particularly in long-term ECMO management, EIC may eventually be complicated by dysregulation of coagulation activation, malfunction of fibrinolysis, consumption coagulopathy, and impairment of anticoagulant systems, leading to a final common pathway of fulminant coagulation failure and the clinical presentation of DIC. The pathophysiology of EIC is likely to be heterogeneous and multifactorial. Acquired von Willebrand syndrome caused by the loss of high-molecular-weight multimers of von Willebrand factor due to high shear stress under ECMO, complications associated with heparin-induced thrombocytopenia and thrombotic microangiopathy, and many other factors can be potential mechanisms of thrombotic and bleeding events during ECMO management.,10 In the current case, COVID-19-associated coagulopathy, inflammation, and endothelial cell dysfunction may have affected the EIC. ECMO circuit changes and withdrawal drastically improved the systemic coagulofibrinolytic activation and refractory oronasal bleeding. These responses demonstrate that prolonged ECMO management without anticoagulation is significantly associated with excessive coagulofibrinolytic activation, and the markers sensitively represented the coagulofibrinolytic fluctuation.The patient’s hemostatic condition under anticoagulant therapy was controlled within the localized thrombus formation without a systemic coagulofibrinolytic response. After the discontinuation of anticoagulation, systemic coagulofibrinolytic activation coexisting with bleeding and thrombotic complications occurred, which was in accordance with the definition of DIC, namely, “an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes,” as proposed by the International Society on Thrombosis and Haemostasis. In the current case, TEG6s® was effective for adequate transfusion of plasma and PLTs from a functional aspect. It should be noted that there are some differences between the VHAs and coagulofibrinolytic markers. VHAs indicate hemostatic functional ability, whereas markers such as TAT, SF, PIC, and FDP represent coagulation and fibrinolysis activity. As described above, we should detect and improve excessive coagulation activation, and in this regard, markers are likely to be able to detect this response more sensitively than VHAs. Actually, in a previous report of cardiovascular surgery, coagulofibrinolytic markers including TAT reflected surgical stress and were associated with bleeding volume. Although UFH could not be started because of the patient’s bleeding tendency, long-term ECMO management was made possible in this case by controlling the excessive activation of coagulation by exchanging circuits at the required time. Considering the social aspects of the COVID-19 epidemic and treatment cost, optimal anticoagulant management for COVID-19-associated coagulopathy, and EIC should be established. Under such circumstances, monitoring VHAs for adequate transfusion of blood products and markers for controlling coagulofibrinolytic activation may be an optional method for the safe and adequate management of ECMO.VHAs are whole-blood point-of-care coagulation assays used to measure the viscoelastic properties of clots. Currently, the use of VHAs is recommended to guide the administration of blood products and coagulation factors in the presence of bleeding. Recent studies have suggested that hypercoagulable states, as demonstrated by VHAs, can predict the risk of thrombotic complications.The patient with severe COVID-19 ARDS presented herein exhibited unique coagulofibrinolytic responses under long-term VV-ECMO management without anticoagulation: abrupt coagulofibrinolytic activation with bleeding and thrombotic complications developed from hemostasis to systemic coagulopathy. Monitoring coagulofibrinolytic status using markers and VHAs may be effective for safe ECMO management in such situations.The authors would like to thank the nursing staff of the Intensive Care Unit 2 at Ehime University Hospital for their assistance.Conceptualization: Hironori Matsumoto, Satoshi Kikuchi, Satoru Murata, Muneaki Ohshita, Yutaka Harima, Suguru Annen, Naoki Mukai, Yuki Nakabayashi, Shirou Ogawa, Mitsuo Okita.Supervision: Norio Sato.Writing – original draft: Hironori Matsumoto.Writing – review & editing: Jun Takeba.


Emerging adulthood concerns the transition from adolescence to adulthood. It foresees the separation from the family and the creation of new significant relationships, whereby specific attachment styles might be triggered when facing these challenges. The present study investigates the influence of retrospective memories associated with childhood separation anxiety symptoms upon emerging adults’ romantic avoidant vs. anxious attachment styles including adult separation anxiety symptoms as mediators. Age and gender were included as covariates. A community sample of N = 394 Italian emerging adults  completed self-report measures. The results showed that the participants presented a greater anxious attachment rather than an avoidant attachment style. Moreover, both adult separation anxiety and the memories of early separation anxiety were positively and significantly associated with anxious attachment and not with avoidant attachment. A mediation model conducted and focused on anxious attachment showed that, although not directly associated, child separation anxiety did show a significant positive indirect effect on anxious attachment as mediated by adult separation anxiety symptoms. Thus, the findings highlighted the influence of adult separation anxiety symptoms and retrospective childhood memories of separation anxiety upon anxious adult romantic attachment, yet not on avoidant attachment style. Clinical implications are discussed and suggestions for future research are provided. Attachment theory has long acknowledged that the need to form and maintain close bonds is fundamental to humans throughout the course of life . Attachm“Emerging Adulthood” is defined as a developmental period ranging approximately from 18 to 30 years of age and refers primarily to young adults who do not have children, do not live in their own home, or do not have sufficient income to become fully independent . AlthougDiagnostic and Statistical Manual of Mental Disorders (DSM-5) [In this regard, it is noteworthy that the fifth edition of the  (DSM-5)  has incl (DSM-5) . Among y (DSM-5) . The lit (DSM-5) . However (DSM-5) . The cha (DSM-5) ,26, corr (DSM-5) , and com (DSM-5) ,28, with (DSM-5) . However (DSM-5) . Researc (DSM-5) ,31. StudNotably, all of the aforementioned literature regarding separation anxiety focuses on the identification of early memories of separation anxiety symptoms and/or adult separation anxiety in clinical samples. Very little attention has been given to community samples whereby an ASAD clinical diagnosis is not considered a milestone ,32. To oThe present study is aimed at exploratively investigating how retrospective memories of separation anxiety symptoms and adult separation anxiety can be associated with emerging adults’ avoidant and anxious attachments. In this regard, the first aim is to investigate which romantic attachment style (anxious vs. avoidant) is prevalent among Italian emerging adults. The second aim is to evaluate how romantic attachment is associated with adult separation anxiety profiles based on the symptom levels observed in adult separation anxiety as well as the retrospective memories of separation anxiety symptoms. Last, the influence of retrospective memories associated with early memories of separation anxiety symptoms upon emerging adults’ avoidant vs. anxious romantic attachment styles, including adult separation anxiety symptoms as mediators, is explored.A total of N = 394 Italian emerging adults participated in the study, of which 276 (70%) were female and aged between 18 and 30 years . The participants were all university students (undergraduate and postgraduate). All participants indicated that they had not been hospitalized with symptoms of psychiatric disorders in the past two years. A strong minority  reported previous psychological counselling or intervention in the past two years for mild problems, unrelated to separation anxiety problems. Only 34 participants (4.1%) were in a common-law relationship and only one (3.6%) was married. The study procedure was conducted in compliance with the Declaration of Helsinki  and approved by the Ethical Interdepartmental Committee of Padova University (1266/2013). The participants provided their consent prior to their participation. All were informed that their data were confidential, that they could refuse to disclose information they did not want to provide, and that they could withdraw from the study at any moment. Trained master’s students assisted the participants in the compilation of the self-report measures during regular university hours.The Separation Anxiety Symptom Inventory (SASI)  is a 15-The Adult Separation Anxiety–27 (ASA-27)  is a 27-The Experiences in Close Relationships Questionnaire-Revised (ECR-R)  is a 36-p < 0.00), indicating that none of the variables presented a normal distribution.The analyses were performed using R and SPSS v.20. The preliminary analysis regarded demographic  and descriptive information  and the assessment of the variables’ normal distribution through the Shapiro-Wilk test. The Shapiro-Wilk test was used to assess the distribution of the present variables . All tests were significant  vs. avoidant (ECR-Avoidance) attachment was compared using the Wilcoxon test for paired samples. Furthermore, to explore the variables’ associative patterns, Spearman rho correlations were performed and interpreted when Cohen’s d effect size was at least medium (>0.30) . All anaA mediational model (PROCESS Model 4)  was perfThe descriptive information is reported in p < 0.00).The Wilcoxon test for paired samples was performed to investigate differences in the participants’ levels of ECR-Anxiety vs. ECR-Avoidance. The results showed that the current sample presented significantly higher scores for ECR-Anxiety compared to those for ECR-Avoidance  as well as the covariates  accounted for the 13.67% of ECR-Anxiety total variance. This model highlighted a significant total effect .In particular, as shown in The present study aimed at exploratively investigating how retrospective memories of separation anxiety symptoms and adult separation anxiety could be associated with emerging adults’ romantic avoidant versus anxious attachment styles. Few studies have investigated these relationships and those that did, were mainly focused on clinical populations ,33. ThusThe results related to the first and second aims of the present study pointed out that emerging adults showed a significantly higher anxious romantic attachment rather than an avoidant romantic attachment style. However, a positive significant correlation was found between anxious and avoidant attachment styles, which can indicate an increase in the use of anxious strategies as well as an increase in the use of avoidant strategies in romantic attachment styles. The results showed that early memories of separation anxiety and separation anxiety symptoms in adulthood are related to a more anxious attachment style. These findings are further supported by the plots showing the linear association between variables, which highlight how emerging adults with anxious romantic attachment are distributed evenly regarding both separation anxiety symptoms and early memories of separation anxiety. This distribution was not observed for avoidant attachment, which has no significant association with separation anxiety symptoms among emerging adults. Therefore, no mediation model was performed considering avoidance attachment as the dependent variable, whereas it was carried out considering anxious attachment and including both adults’ reported separation anxiety symptoms and early memories of separation anxiety. This latter mediational model, as the third aim of the present study, shows that early memories of separation anxiety symptoms influence anxious romantic attachment, only as a contribution to the presence of separation anxiety symptoms. In other words, the more that young adults report early memories of separation anxiety and report having experienced adult separation anxiety symptoms, the more they display an anxious attachment style in their close relationships. These results are consistent with the available literature regarding the developmental challenges that emerging adults have to face, characterized, in particular, by the experience of “feeling in-between” . EmerginExtensive research has explored the impact of early experiences, particularly within the family ,46, upon


Design of the next-generation of therapeutics, biosensors,andmolecular tools for basic research requires that we bring proteinactivity under control. Each protein has unique properties, and therefore,it is critical to tailor the current techniques to develop new regulatorymethods and regulate new proteins of interest (POIs). This perspectivegives an overview of the widely used stimuli and synthetic and naturalmethods for conditional regulation of proteins. A systematic control of proteinfunction using various chemical and biochemical strategies is calledprotein regulation.3 Proteins are saidto be conditionally regulated if these strategies are executed inresponse to a specific stimulus.Protein regulation orchestrates nearlyall molecular events oflife. Its importance can be realized from the existence of about sixmillion proteoforms which are produced from just ∼20000 humangenes.Biochemical reactions are largelyperformed in complex milieu ratherthan in isolation. Consequently, any participating species from reactionsoccurring in proximity can influence the reaction of interest. Therefore,life cannot persist if orthogonal mechanisms are not in place to regulateproteins in both the spatial and temporal dimensions. Understandingprotein regulation is of utmost importance for chemists and biologists,and this review investigates the widely used techniques for proteinregulation in biochemical research.4 In contrast,chemical strategies allow the study of proteins that are otherwiseintractable to genetic techniques, such as proteins that are essentialin germline and are impossible to knock out or down with genetic manipulation.Chemical methods also achieve regulation at a faster rate as theyaffect protein activity at the post-translational level and cost lessin comparison with tedious genetic engineering techniques.5 Some protein regulatory strategies also makeuse of a combination of chemical and molecular techniques, which arealso discussed in this review.Several molecular techniquestarget protein activity at transcriptionalor translational levels. While they have several advantages, theyalso have limitations and cannot be used to regulate all POIs, suchas those required for viability.This perspective aims to providean overview of different approachesacting at the post-translational level, rather than extensively reviewingeach strategy. Conditional protein regulation can be systematicallystudied by understanding (a) the stimulus and (b) the method of regulation1. ProteiBroadly, both physical and chemicalagents are known to regulateproteins. They generally induce changes in the protein by either makingor breaking bonds, or changing its conformation or the nature of itsinteractions.7 By understanding thelight-induced chemical and structural changes in such proteins whichare discussed later in this perspective, several light-responsivemoieties were developed to be incorporated into POIs to trigger structuraland conformational changes on demand. Most light responsive elementsin use respond to visible-NIR light as UV light (a) is toxic to nucleicacids and (b) cannot be used for in vivo applicationsdue to low tissue penetration.8In nature, light is responsible forthe regulation of photoreceptor proteins which participate in visualperception and phototaxis.10 Both the pressure- and thermo-receptorsbelong to the same family of proteins, and they change their conformationto permit the movement of cations across cell membranes to convertmechanical forces into electrochemical signals. Sensing and respondingto changes in temperature, blood pressure, touch etc. is typicallyaccomplished by proteins responding to force.14 So far, temperature and pressurehave been used less frequently for synthetic regulation of proteins.Pressure,sound, and temperature are other physical stimuli for post-translationalprotein regulation.17 Small molecules act as stimuli at the transcriptional, translational,and post-translational levels, of which only those acting on proteinswill be discussed in this text. Though biomolecules act as stimuliwith higher specificity, they have short half-lives in plasma andcannot easily pass through membranes.18 Other noteworthy chemical stimuli include chemical gradients inthe environment. For instance, pH gradients can be observed betweendifferent tissues or intracellular organelles which affect the protonationstates and protein conformations and hence modulate their activity.21 Oxygen gradients affect proteins such as the hypoxia inducible factor,which is hydroxylated at prolines at high oxygen concentrations fordegradation.24 Reactive oxygen species (ROS) act as stimuli forredox-switch proteins which may be regulated through chemistry attheir reactive centers including thiols or metals.25 Synthetic hypoxia- and ROS-sensitive immolative moietieshave also been developed for protein regulation.27 Finally, biomolecules themselves act as stimuli for protein regulation.28Chemical stimuli are responsible forthe regulation of a significant proportion of proteins. Several chemicalsranging from ions to small- and biomolecules, which include lipids,DNA, RNA, and even other proteins, can act as stimuli.A stimulus will guide the POI intoeither one of the paths leadingto activation or inactivation. These paths or regulatory strategiescan be studied by categorizing them as discussed in this text. Someproteins are also known to be regulated using a combination of thesestrategies.Many stimuli interact noncovalently with POIs byassociation or dissociation. Such interactions lead to changes inthe protein interactome, conformations, and creation or destructionof enzymatic active sites to control biochemical activity.31 Biologically, feedback inhibition of metabolic pathways occurs throughnoncovalent interactions.32 Sensor proteins,such as calmodulin and GTPases, are also regulated by conformationalcontrol. Additionally, it is important to note another type of allostery,viz., cooperativity which permits graded control of protein activity.Binding of oxygen at allosteric sites on hemoglobin is a well-studiedexample of cooperativity.33 The abilityof small molecules to control protein complexation has been used inthe pharmaceutical industry for therapeutic benefit and to controlthe delivery of biotherapeutics.36 If any POI is inerttoward the action of a stimulus, a frequently employed strategy isto fuse the POI with a domain which recognizes a small molecule throughnoncovalent interactions.38Themajority of pharmaceutical agents fall under this category 2a. It is42 For instance, the immunosuppressant drug tacrolimus (FK-506) bindswith a ubiquitously expressed 12 kDa protein called FKBP12.43 This interaction is very specific and thereforedimers of such compounds are used to induce protein interactions.46 Overall, this process was named chemically induced dimerization(CID), and was applied to study the effects of protein associationsin living systems such as Ras signaling, Fas signaling, and T-cellreceptor activation in lymphocytes, etc.48 CID did not just aid in making fundamental discoveries, but alsowas developed as a tool for the clinics to control the side effectsof CAR T cell immunotherapy, and as a switch control in patients thatdevelop Graft Versus Host Disease after T cell transplantation.51 CID is generally made possible with any POI by fusing it with bindingdomains genetically. Since the dimerizing molecule brings two fusionproteins together by binding with the fused domain, interactions ofthe POI can be studied without perturbing its chemistry or conformation.These bivalent ligands have been further modified to prevent theirinteractions with POIs until they encounter a specific stimulus. Thisadditional layer of regulation made spatiotemporal control of CIDpossible by modifying the bivalent ligands to be sensitive to specificstimuli.54Many small molecules and drugs are also capableof inducing proteinassociations 2b.39−42 55 Whilethese proteins naturally participate in cellular activities like phototropismand circadian rhythms, they are now extensively used by chemical biologiststo study activities dependent on protein density. LOV and CRY2 domainsrequire flavin cofactor  and respondto blue light. In contrast, PHYB requires the cofactor phycocyanobilinwhich is not available in animal systems. Therefore, phycocyanobilinmust be included in those assay systems which use PHYB domains. Thenatural LOV domain comprises of a smaller cysteine containing “Per-Arnt-Sim”or PAS domain bound to the flavin cofactor. The cysteine in the PASdomain reacts with flavin covalently when exposed to light, leadingto changes in conformation and unfolding of a specific α-helixin the LOV protein. The POI can be caged by fusing it with this α-helixand uncaged using light. This α-helix may also be engineeredto bind with the POI and control protein–protein interactions.Furthermore, the LOV domain has been engineered to produce a varietyof light-induced effects which have been reviewed extensively.56 Briefly, they can be used for controlling proteinlocalization (optogenetic systems iLID and TULIP), producing homodimers(TAEL and Vivid) or heterodimers (Magnets). Light controlled dimerization(or multimerization) is also possible with another fluorescent protein,Dronpa (K145N mutant), which remains as a monomer in dark (∼500nm). It associates into a tetramer when exposed to high energy light(∼400 nm). This property of the protein was utilized to controlPOIs. The association of a POI was controlled with light by expressingit as a fusion protein with with the Dronpa K145N domain on the N-or C-termini.58 Like CID, LAD using fusion proteinscan be used to study protein associations without introducing conformationalchanges in the POI.Certain proteins such as the light-oxygen-voltage-sensingdomains(LOV domains), chryptochromes (CRY2), and phytochromes (PHYB) changetheir conformations when exposed to light and undergo dimerizationor oligomerization with their specific binding partners .59Obvious disadvantages for both CID and LADmust be noted. In additionto the use of genetic methods to incorporate these domains into thePOI, fusion proteins do not accurately possess the properties of thenatural POI. These domains are usually bulky and change the molecularweights, isoelectric points, and therefore, possibly the localizationor interactions of the POI. Therefore, fusion proteins must be cautiouslyused only if the advantages of the study outweigh the disadvantagesassociated with the system.enzymes ; however, they are structurally different fromPROTACs as they do not contain a linker connecting two independentligands.64 They are single-unit entitieswith the capacity to establish new and stable interactions betweenthe POI and the E3-ligase.67 Macroautophagy degradation targetingchimeras (MADTACs) including autophagy-targeting chimeras (AUTACs)and autophagosome-tethering compounds (ATTECs) utilize the mechanismsof autophagy to degrade the POI.68 Thebinder in the AUTACs is covalently linked with a guanylated cysteine,a moiety mimicking the protein post-translational modification S-guanylation.Proteins with S-guanylation are known to undergo degradation throughautophagy via an unknown mechanism and AUTACs install this labelingspecifically on the POI through directed and noncovalent interactions.69 AUTOTACs are a different class of bifunctionalmolecules that act intracellularly by binding with the POI on oneterminus and p62 (SQSTM1) on the other. AUTOTAC induced associationof the POI and p62 leads to the activation of autophagy pathways anddegradation of the POI.70 ATTECs are originallymolecular glues that interact with the POI and the autophagosome membraneprotein LC3. The POI and LC3 interact through the interactions madeby the ATTEC and are internalized into the autophagosome for degradation.71 Deubiquitinase-targeting chimeras (DUBTACs)mediate interactions between the POI and the deubiquitinase enzyme,which results in the removal of ubiquitin labels thereby preventingthe degradation and increasing the half-life of the POI.72 Phosphorylation-inducing chimeric small molecules(PHICS) are bivalent molecules which bind with and bring togethera kinase (either AMPK or PKC so far) and the POI to enable phosphoryltransfer on to the POI.73 Acetylation taggers(AceTAGs) and phosphorylation targeting chimeras (PhosTACs) do notdirectly bring the enzyme and POI together but make use of a FKBP12F36V domain genetically installed on lysine acetyl transferaseand Ser/Thr phosphatase enzymes respectively to mediate any associationsbetween them.75Otherbi- or multivalent ligands are also designed to induce associationsspecifically between two or more POIs and enzymes 2b. Thep77 In addition to transmembrane POIs, both these bivalent ligands arecapable of targeting extracellular proteins for degradation via interactionswith the M6P receptor, followed by lysosomal degradation. Antibody-basedPROTACs (AbTACs) and Proteolysis Targeting Antibodies (PROTABs) arebispecific IgG antibodies which have the arms engineered to bind with(a) a POI and (b) a transmembrane E3 ligase like RNF43 or ZNRF3.79 Both PROTABs and AbTACs were observed to ubiquitinate the POI resultingin its endocytosis and/or degradation. GlueTACs are engineered nanobodiesconsisting of (a) a proximity reactive noncanonical amino acid onits paratope and (b) a cell-penetrating peptide and lysosomal sortingsequence (CPP-LSS) away from the binding site. GlueTACs covalentlylabel the membrane POI using the noncanonical residue, and are internalizedinto the lysosome for degradation due to the presence of the CPP-LSSmoiety.80 Cytokine receptor-targeting chimeras(KineTACs) are engineered antibodies, like PROTABs, that also havetwo arms which bind with the POI (extracellular/membrane) and thecytokine receptor, respectively. Upon association, the POI-KineTAC-cytokinereceptor complex is internalized into lysosomes for degradation.81Transmembrane proteins,which include ion channels, molecular receptors,adhesins etc., constitute a significant proportion of the functionalproteome and can be important targets in drug discovery. Conditionalcontrol of transmembrane POIs thus enables the development of newtherapies. However, design of bifunctional molecules for this purposeis a challenging task as they generally are incapable of bringingthe transmembrane POI and any cytosolic enzyme closer in space asthe majority of the POI is embedded in lipid layers, thus making itless accessible. Strategies which have been successful so far targetthese POIs on their extracellular domains. Molecular degraders ofextracellular proteins through the asialoglycoprotein receptor (MoDE-As)and lysosome-targeting chimeras (LYTACs) are generally mannoslyatedand bring the membrane POI and mannose-6-phosphate (M6P) receptortogether, whose association is followed by internalization of thereceptor-POI complex into a lysosome for degradation.83Unlike the small molecules discussed abovewhich aid in the associationof proteins, protein–protein interaction modulators (PPI modulators)prevent such protein–protein interactions (PPIs) by bindingnoncovalently at either of the protein interfaces aiding in theirdissociation. The interfaces involved in the interactions are generallylarge in comparison with the active or allosteric binding site ofa small molecule on a protein or an enzyme. Moreover, these interfacesare flat and the majority contain hydrophobic residues thus makingan efficient PPI modulator design very difficult.85Split inteins are typical examples of protein activation uponassociation2c. The P87 Complementation has also been applied to improve the therapeuticpotential of recombinant cytokines, the use of which is limited inthe clinics due to severe side effects. A two-component strategy wasdeveloped that requires localization that can be independently targetedto restrict activity to cells expressing two surface markers.88 The association of the two components regeneratesNeoleukin-2/15, a IL-2/15 mimetic designed for both trans-activating immune cells surrounding the target cancer cells and cis-activating to directly target immune cells.89 This technique is also successfully used formitochondrial genome editing, specifically deamination of cytidinesin dsDNA using DddA-derived cytosine base editor (DdCBE). This proteinis an interbacterial toxin which potentially has toxicity. To lowerthis toxicity and off target reactions, DdCBE is engineered into splitinactive protein domains. Cytidine deamination is initiated only bythe fusion of the two domains near the target DNA.90 Nanobody-fused split O-GlcNAcase  are enzymes that remove the O-GlcNAc post-translationalmodification from the POI. This protein is engineered into split domainsto reduce its off-target activity, thus using complementation to improvethe selectivity of the enzyme toward the POI.91 Complementation also is shown to be effective for improving proteomicsstudies. Split-TurboID cannot biotinylate proteins until the domainsassociate in the presence of rapamycin (CID). The resolution of massspectrometry-based proteomics when TurboID complementation was achievedwithin specific organelles was improved, resulting in the identificationof new proteins.92Complementationis another strategy typically requiring noncovalentassociations for protein activation 2d. In th93 This leads to dimerization; other possibilitiesof substitutions  are also possible leading to the formation of oligomers andsometimes, protein fibrils.94 This techniquehas been synthetically applied to POIs to regulate their activitiesunder the influence of specific stimuli.96Domain swappingis generally applied to produce dimers or oligomersof a POI 3a.Here,98 However, a few methods have beendeveloped for specific proteins; but they may not be applied to anyPOI. Histidine residues are well studied and used for achieving pHresponsive conformational control by incorporation at specific sitesin proteins; their participation in protein folding through intramolecularhydrogen bonds can be controlled with pH as their protonation at anypH below ∼6.5 will result in the loss of hydrogen bonds, ultimatelyleading to large changes in protein conformation .Kinases and phosphatases catalyze the addition and removal of phosphategroups respectively from their target proteins. This process generallyoccurs on serine, threonine, and tyrosine residues on the POI. Thistype of regulation is so widely used that about 3% of the proteinsin yeast are kinases and phosphatases. It must also be highlightedthat this method of regulation is chosen to control the activity ofnumerous classes of proteins including ion channels, membrane andstructural proteins, enzymes etc.33 SeveralPTMs like ubiquitination, performed by ubiquitinases and deubiquitinases,are targeted toward lysine residues on different proteins.107 Besides labeling the protein for degradation,ubiquitination is also used as a strategy to control protein localizationand interactions.108 Additionally, thepost-translational covalent modifications on histones too are greatlystudied to understand epigenetics and gene expression.109Oneof the most widely employed ways to regulate proteins irreversible approachfor protein regulation. Proteolysis itself can also be regulated byother proteases; the pancreas produces inactivated enzymes calledzymogens, which are activated in the small intestine by other specificproteases. Most protein hormones are first created as pro-hormonesand are activated into hormones by proteolytic activity.110 Viruses also express pro-proteins, which mustbe hydrolyzed by proteases to form functional proteins as in the caseof HIV.111 Critical processes such as bloodclotting occur only through the proteolytic activation of the coagulationcascade.112 One of the applications ofthis irreversible covalent chemistry is the development of enzyme-activabletherapeutics. Bulky moieties are introduced on the POI to preserveit in an inert state until it encounters an enzyme that cleaves offthese moieties to activate the POI.117 Antibodies are also regulated in a similarfashion by introducing cleavable bulky moieties on the paratope  are strategies whichare widely used for this purpose. Cyclization restricts peptides fromattaining a specific conformation for recognition by proteases, andPEGylation prevents any POI-protease interactions.122Proteolytic action is an paratope 4a.118−12123 These covalent allosteric inhibitorsare generally irreversible; they trap the POI in a specific conformationfor a long duration via a two-step process. The first event is relativelyfast and involves the binding of the small molecule at the allostericsite, followed by a second slow event which involves the covalentmodification of the target protein.124 Anyallosteric interaction ultimately alters protein activity by inducingconformational changes. Design of covalent allosteric inhibitors asdrugs is a challenging task and they can cross-react with other homologousproteins resulting in adverse effects. On the other hand, it is observedthat drug-resistance is not acquired with covalent allosteric inhibitors.125Many small molecules react covalently with proteinsat allostericsites.128A significant proportion of covalentinhibitors are directed towardthe active site of an enzyme. The small molecules have other chemicalgroups such as hydrogen bond donors/acceptors in addition to the reactivefunctionality to increase their specificity toward a particular enzyme.Suicide inhibitors also bind at the active sites of the enzyme andmake covalent connections during the catalysis, thus rendering theenzyme inactive. Both these types of active site-directed covalentinhibitors affect the activity of an enzyme by preventing the entryand catalysis of its substrate.132 Alternatively, bifunctional molecules likeglutaraldehyde or bis-acids (EDC/NHS chemistry) may be used to cross-linkproteins on their basic residues like lysine nonspecifically.134 The latter approach, though very efficient, may ultimately inactivatethe protein. In the context of vaccine design, glutaraldehyde-basedcross-linking was used to stabilize the HIV envelope glycoproteinBG505 to improve neutralizing antibodies against HIV.135Chemical cross-linkingis another strategy to regulate proteins.This is achieved through bioconjugate chemistry, or bioorthogonalreactions or enzymatic labeling as in the case of protein–protein(homo- or hetero-) dimers.136 However, bycombining an intein domain with sensing and reporter domains, therate of protein splicing activity can be controlled by external stimulileading to the emergence of conditional protein splicing (CPS).138 Numerous examples of CPS have been reported with small molecules,light, temperature, and pH.140 Poor solubility, localenvironment optimization and limited reversibility are typical problemsin the development of CPS as a generic method.Unlike the split inteins discussed earlier,inteins are fully hostedby the protein from which they autocatalytically excise themselves.The chemistry of excision is the same as seen in split inteins. Proteinsplicing has predominately been utilized in the areas of protein purificationand protein cyclization.148 Unnatural amino acids (UAAs) present bioorthogonal functionalitiesfor easy and site-specific incorporation of caging groups. Alternatively,caged residues can be directly introduced at desired sites in thePOI using genetic code expansion (GCE) and specific stimuli can beused to decage these residues on demand.153 UAAs were also introduced in proteins to permit cleavage of thepeptide backbone at specific sites with an appropriate stimulus.154 A recent advancement in GCE for conditionalprotein regulation is CAGE-prox . This approach relies on computationaltools to identify a key residue in proximity to the active site, whichcan be replaced with a caged residue to conditionally prevent substratesfrom entering the active site.155 GCE alsoallows incorporation of diazirine and tetrazole based UAAs  for photo-cross-linking to study protein–proteininteractions. These proteins can then be unlinked using hydrogen peroxide.158Protein activitycan be temporarily turned off by caging the POIusing site- or ligand-specific chemistry 4b. The e161 Enzymes have been successfully regulated using this strategy bytethering them with an active- or allosteric-site binder.164 Both natural and unnatural polymers have been used to constructthe tethers.In tethered pharmacology, proteins are regulated by conjugatinga tether to the POI which generally terminates with a small- or biomoleculethat binds weakly with the POI, thereby inhibiting its activity 4c. TetheIn thisapproach, the POI is generally not modified or influenced by an effectorto modulate its conformation. Activity is only controlled by removingor concentrating the POI at the site of action. The compartment mayor may not be a physical enclosure. Such approaches are discussedbelow 5.165 Antibody-protein conjugatesor fusion proteins are generally used for extracellular tissue targeteddelivery. Comparatively, intracellular targeting of proteins is furtherchallenging as it requires translocation across (multiple) biologicalmembranes. While a few strategies have been reported, there is stillneed for a robust generic method for delivering intact proteins acrossmembranes.168 Nano-OGT/OGA (nanobody fused O-GlcNAc transferase/O-GlcNAcase)are enzyme-nanobody constructs which can be used to install/removeO-GlcNAc modifications on selective proteins. The nanobody helps directthe enzyme toward the POI, thereby increasing the effective concentrationsof the enzyme and POI for improved biocatalysis.169Protein activity may be directed toward specificsites withoutthe use of physical enclosures such as membranes. This is typicallyachieved by linking the POI with antibodies or lectins that bind withspecific biomolecules expressed exclusively by the respective compartmentsin the body.170 Amphiphile-mediated depalmitoylation,on the other hand, aids in the removal of lipid modifications fromproteins. Specifically, a cysteine containing amphiphile is observedto react with the thioester linkage of a S-palmitoylated membranebound protein, thus unbinding the POI from the membrane via a nativechemical ligation-like reaction.171 GCE(discussed earlier) was also applied to control post-translationalmodification of RanGAP1, the POI in this study and its localizationat the nuclear membrane. Incorporation of a lysine protected withan aryl-azido group (PABK) prevented SUMOylation of the POI. Decagingthe lysine with 2-(diphenylphosphanyl)benzamide localized the POIat the nuclear membrane through interactions with the nuclear porecomplex.172 Magnetic control of proteinscan also be achieved by conjugating or fusing the POI with an antibody-functionalizedwith superparamagnetic nanoparticles.173 Alternatively, a functionalized antibody can be directly used tobring together the POI in the cell through noncovalent interactions,whose localization can be controlled using an electromagnetic needle.174 The anchor away system is another techniqueused to evacuate nuclear POIs conditionally and rapidly away fromthe nucleus using specific chemical stimuli to tether them in thecytoplasm resulting in their loss of function. This loss of functionis reversible and can be restored when the chemical stimulus is removed.Multiple nuclear proteins can be regulated simultaneously using thissystem.176 The PhyB and LOV2 systems, described underlight induced dimerization, have also been used to localize proteinsintracellularly with blue or NIR light.177Alternative chemical methods also permit localization of proteinsat desired sites without a physical enclosure. Lipidation is a popularapproach in which the POI is covalently attached with lipid anchorsto direct a protein to particular membrane sites. One prominent chemicallipidation approach uses a photoinduced tetrazole-alkene cycloadditionreaction to install exogenous lipid dipolarophiles onto proteins inlive cells.179 Althoughfew studies show that proteins lose their integrity when encapsulatedin lipid membranes, some have encapsulated peptide- and protein-therapeuticsto explore their capacity for delivery.183 Furthermore, these membranes are functionalized for targeted therapyor membrane permeation.185 Both synthetic andprotein-based cages have been used to encapsulate POIs.187Encapsulation of POIs in membranes or cages is an effectivewayof spatially controlling protein activity. POIs have been encapsulatedby complex coacervation with poly ionic peptides or compounds to improvetheir stability in circulation.189 LLPS can either lead to reduction in protein activity as it is removedfrom the reaction mixture or improvement of the same due to increasedconcentration within the excluded compartment. One of the major requirementsfor a protein undergoing LLPS is the presence of a charged intrinsicallydisordered domain (IDD), which provides the POI a high conformationalentropy and freedom to explore a variety of conformations.191 IDDs generally contain many residues. While peptides containing6–18 residues were observed to induce phase separation whenincorporated at the C-termini of specific POIs, short peptides generallycannot induce LLPS for any POI as they do not contain the structuralfeatures discussed above.192 Both experimentaland computational attempts are being made to predict the structure–LLPSrelationships of peptides.197 Synthetic protein-recruiting/-releasing condensates (SPRECs) aredesigned which are capable of sequestering POIs, as well as releasingthem in living systems. Reversible POI sequestering was achieved bycontrolling its associations with the phase separating protein usingthe LOV systems discussed previously for light induced associationand dissociation, respectively.198 Molecularbiology techniques are currently being used to construct fusion proteinsof any POI with an IDD to allow controlled phase separation. Stimulisuch as light and small molecules are being explored for conditionalregulation.201 Though LLPS is a powerful tool for proteinregulation, it should be noted that this process can initiate proteinaggregation and misfolding, thus leading to many problems. A successfulstrategy for conditional regulation must be reversible and maintainprotein quality after LLPS.Some proteins are also regulated by liquid–liquid phaseseparation (LLPS), initiated by both chemical and physical stimuli.203 Preclinical studies have repeatedly shown that conditionally regulatedproteins are superior to their native forms. However, most biotechnologicalproducts in clinics are not conditionally regulated; instead, nativeprotein forms are used as therapeutics. There is a need for the developmentof robust methods such that they will be translated into clinicalsettings and help the development of safer and improved biotherapeutics.Protein regulation is not just centralto life, but also to themajority of fundamental discoveries, preclinical research and therapeuticdesign. With rapid advancement in the fields of protein engineeringand bioconjugation, new proteins are constantly developed which requireintelligent methods of regulation. The recent discovery of a NOS bridgein proteins as a redox switch is another reminder that the field ofprotein regulation is vastly unexplored.Biological methods are not cost-effective; however they are genericand reliable. For instance, small and easy design modifications torespective plasmids can allow knock of any DNA sequence using CRISPR.Therefore, they are widely used by both chemists and biologists. Sofar, very few generic chemical tools have been developed, which tooare difficult to be translated for regulating other POIs. Moreover,the majority of the techniques require the use of biological procedureslike protein engineering or expression as discussed in this perspective.We believe that it is for this reason chemical approaches  are not appreciated enough for translational research yet.In situ biosynthesis of large proteins andnucleic acids, which are extremely crucial for biocatalysis and molecularrecognition, makes the development of biological tools easier in comparison.On the other hand, chemical methods must overcome the challenges inboth the synthesis and delivery of such complex biochemical componentsto the target site. This limits the rate of development of chemicalapproaches for conditional protein regulation.Biological tools have also become successful because of the utilizationof the host’s biosynthetic machinery to obtain the desiredeffect. The goal of achemical regulatory method is to achieve the desiredbiological effect on a specific POI. This can be achieved using site-and residue-specific chemistry which is bioorthogonal to any otherprocesses using water-soluble and readily activated reagents. Therefore,the growth of this technology, i.e., conditional protein regulation,also depends on how fast the progress is in the field of bioconjugation.Chemists are now providing elegant and inexpensive solutions to thisproblem. To overcome the problems of molecular recognition and reactivityfor instance, they are making use of residue specific reagents incombination with targeting moieties such as antibodies or ligandsidentified via high throughput screening and mass spectrometry. Weanticipate that more chemical methods will be developed in the comingyears with the expansion of our bioconjugate chemistry toolbox. Inaddition, there is vast space for the evolution of new chemistry withthe discovery of novel biochemical regulatory processes. Such newmethods of regulation may broadly fall under one of the categoriesdiscussed in this review. We hope that this perspective serves asa guide for those within and outside of the community and inspiresthe next generation of researchers to take up these challenges.


Mucinous ovarian carcinoma is a rare subtype of epithelial ovarian cancer. Despite being a chemoresistant tumour type, surgical resection and chemotherapy are still the current standard for management. This narrative review aims to explore the current evidence for targeted therapies in mucinous ovarian carcinoma. A review of the literature was performed to identify clinical trials and case reports of targeted therapy in patients with mucinous ovarian carcinoma. The databases and registers  were searched for articles published between January 2009 to June 2021 using keywords specific for mucinous ovarian carcinoma and targeted therapy. Records were screened and assessed for eligibility based on inclusion and exclusion criteria. From 684 records, 21 studies met the criteria to be included in the review. A total of 11 different targeted therapies were identified, each demonstrating varying degrees of clinical evidence supporting further investigation in patients with mucinous ovarian carcinoma. Targeted therapies identified in this review that warrant further investigations are bevacizumab, trastuzumab, nintedanib, AZD1775, sunitinib, cediranib and pazopanib. Many of the therapeutic agents may be investigated further in combination with other targeted therapies or chemotherapy. More clinical trials focusing on targeted therapy specifically in patients with mucinous ovarian cancer are required to inform clinical use. Multinational efforts are likely to be required to successfully conduct trials in this rare tumor type. Mucinous ovarian carcinoma accounts for 3–5% of all epithelial ovarian cancers.KRAS/NRAS mutations (65.8%), TP53 mutation (65.2%), ERBB2 amplification (26.7%) and BRAF mutation (8.7%).BRCA1/2-mutated tumors. The highly specific nature of targeted therapy suggests that these agents are likely to use mechanisms of action that evade the chemoresistant features of mucinous ovarian carcinoma cells. Examples of targeted agents include monoclonal antibodies or small molecules that target specific tumour-associated antigens, tumour-promoting molecules or against immune checkpoint proteins. Therapeutic agents targeting specific biomarkers and key molecular drivers of mucinous ovarian carcinoma such as ERBB2 and KRAS may exhibit promising results.Targeted therapy involves agents that target cancer-associated molecules to specifically inhibit the growth and spread of malignant cells. In contrast, chemotherapy uses cytotoxic drugs that kill both cancer cells and normal cells, hence, patients are likely to experience more toxic adverse effects.ation 26.% and BRAThis narrative review aims to evaluate the literature to identify and appraise the outcomes of clinical trials of targeted therapy that have included patients diagnosed with advanced (stage II-IV) or recurrent mucinous ovarian carcinoma. Currently, clinical trials of targeted therapy specifically for mucinous ovarian carcinoma have not been completed due to the rarity of the cancer. For instance, the rare tumor trial GOG-0241 evaluating four treatment regimens for mucinous ovarian carcinoma was terminated early due to poor accrual.The principle mechanism of action of monoclonal VEGF antibodies is via the inhibition of tumor angiogenesis. In four phase III ovarian cancer trials, including a total of 42 patients with mucinous ovarian carcinoma, the addition of bevacizumab to chemotherapy significantly improved overall progression-free survival by 2–4 months and median overall survival by 5 months.An alternative mechanism to inhibit VEGF receptors is by the small molecule inhibitor cediranib, which similarly functions by suppressing tumor angiogenesis. The phase III trial ICON6 included 4 patients with mucinous ovarian carcinoma and showed a prolongation in progression-free survival – 11 months in the group that received chemotherapy followed by maintenance cediranib, compared with 8.7 months for chemotherapy only.Platelet derived growth factor (PDGF), similar to VEGF, plays a major role in tumor angiogenesis and tumor cell proliferation. Sunitinib targets multiple receptor tyrosine kinases including the receptors for PDGF and VEGF, thereby inhibiting tumor vascularization and promoting tumor cell apoptosis. In a phase II trial comparing continuous and non-continuous monotherapy with sunitinib in platinum resistant ovarian cancer, the non-continuous regimen demonstrated the best reported response according to RECIST criteria. The response rate  for the non-continuous treatment arm was 15.7%, compared with 5.4% for the continuous treatment arm.In a phase III trial including 37 patients with mucinous ovarian carcinoma, treatment with nintedanib in combination with carboplatin and paclitaxel across all included subtypes resulted in a progression-free survival of 17.6 months with nintedanib and 16.6 months with placebo.A third inhibitor, pazopanib, is effective in treating renal cell carcinoma. The phase III trial AGO-OVAR16 comparing pazopanib in combination with chemotherapy vs chemotherapy alone demonstrated an improvement in progression-free survival. However, this did not translate to an improvement in median overall survival.HER2, also known as ERBB2) gene amplification is shown to drive tumor development and progression in 18–26% of patients with mucinous ovarian carcinoma.ERBB2 gene amplification. After ceasing treatment on experiencing severe adverse effects including peripheral neuropathy, radiographic progression of disease with extensive metastasis was observed 12 months after the last treatment.Human receptor epidermal growth factor 2  inhibition by erlotinib blocks tumor cell growth and initiates apoptosis in EGFR-overexpressing tumor cells.TP53 mutated ovarian tumors demonstrated an objective response rate of about 43%.AZD1775 is an inhibitor of WEE1, which is a tyrosine kinase that regulates cell cycle progression. Inhibiting WEE1 increases the apoptotic response to DNA damage in p53 deficient tumor cells, thereby enhancing the anti-tumor activity of chemotherapy in platinum-resistant tumors. A phase II study of chemotherapy and AZD1775 in CA-125 is expressed by a significant proportion of epithelial ovarian cancers and elevated levels are associated with platinum resistance and increased tumor invasiveness. Abagovomab is a monoclonal antibody that mimics CA-125 and has been shown to induce tumor specific immune responses in preclinical and phase I/II studies. In a phase III trial comparing abagovomab against a placebo as maintenance therapy, 9 patients with mucinous ovarian carcinoma were included. Despite having strongly supporting phase II data, this trial observed no benefit across all patients in recurrence-free survival or overall survival.SRC family kinases and receptor tyrosine kinases are involved in signaling that promotes cell survival, growth, metastasis and angiogenesis.KRAS mutation in mucinous ovarian carcinoma, it can result in proteins that promote uncontrolled cell growth and proliferation. The anti-tumor activity of lonafarnib, a farnesyltransferase inhibitor, is attributed to the inhibition of post-atranslational modifications that are required for Ras to function.Farnesyltransferase is an enzyme involved in the functioning of Ras family proteins, which are critical oncogenes involved in cell proliferation. If a Ras protein is inappropriately activated, as in the case of The main limitations of the clinical trials included in this review are primarily a result of the rarity of mucinous ovarian carcinoma. There have been no completed clinical trials of targeted therapy to date that are specific to patients with this histotype, and only a single trial (GOG-0241) of this nature has been initiated to date before being terminated. Therefore, this review identified studies that have included patients with mucinous ovarian carcinoma. However, the sample size of patients with this disease within the discussed pan-ovarian clinical trials have been relatively low, with the maximum number of mucinous histotype patients in a single trial being 40, in the AGO-OVAR16 trial of pazopanib. The majority of the articles did not provide a histological subtype-based analysis of responses to therapy, limiting the inferences that can be drawn specifically regarding the efficacy of a therapeutic agent in patients with mucinous ovarian carcinoma. Second, in the future targeted therapies are likely to have the potential to be offered to patients with mucinous ovarian carcinoma based on tumor biomarker stratification. Except for trials and case reports in trastuzumab, AZD1775 and erlotinib, many of the trials discussed in this review did not have specific tumor biomarkers determining entry of patients into trials.The main limitations of the review methodology includes the inability to search the full-text or supplementary files for keywords in the databases and registers searched for this review to increase the specificity of the search. This made it quite laborious to look through the baseline characteristics table of trials when screening records. This limitation also makes it likely that trials were potentially missed by the search process utilized in this review – trials that included patients with mucinous ovarian carcinoma but only mentioned this elsewhere in the full-text or in the supplementary materials. Unfortunately, performing such a search using a full-text searching tool such as Google Scholar results in tens of thousands of results, which is unfeasible to screen. Utilizing natural language processing systems may be an avenue for improving search efficacy and specificity in future reviews.The aim of this review was to identify and evaluate the current evidence for targeted therapy in patients with mucinous ovarian carcinoma to inform directions for further investigation of therapeutic agents, thereby contributing to the development of personalized treatment and perhaps potential integration into standard treatment for this disease . DespiteSuggestions for future trials investigating the agents that show promising patient outcomes include aims to optimize timing and duration of treatment and identify potential patient/tumor factors that may predict treatment efficacy. It is likely that multinational efforts will be required to successfully conduct these rare tumor trials in mucinous ovarian carcinoma. However, given the phase III GOG-0241 trial that was terminated due to poor accrual, such trials may never be undertaken. In which case, evaluating outcomes of pan-ovarian or pan-cancer trials in reviews like this may help clinicians make informed choices regarding off-label treatment in mucinous ovarian carcinoma. In the future, prospective registry trials, adaptive trials and basket trials such as Bouquet-ENGOT-gyn2, as well as non-trial options such as multi-institutional series or regional/national databases, could play a role in gathering therapeutic evidence for such rare tumors. Analysis of existing data is challenging given variable reporting mechanisms, missing data and uncontrolled confounders, but could provide stimulus to pursue a promising therapy in a more controlled fashion.


The impact of extreme heat on crop yields is an increasingly pressing issue given anthropogenic climate warming. However, some of the physical mechanisms involved in these impacts remain unclear, impeding adaptation-relevant insight and reliable projections of future climate impacts on crops. Here, using a multiple regression model based on observational data, we show that while extreme dry heat steeply reduced U.S. corn and soy yields, humid heat extremes had insignificant impacts and even boosted yields in some areas, despite having comparably high dry-bulb temperatures as their dry heat counterparts. This result suggests that conflating dry and humid heat extremes may lead to underestimated crop yield sensitivities to extreme dry heat. Rainfall tends to precede humid but not dry heat extremes, suggesting that multivariate weather sequences play a role in these crop responses. Our results provide evidence that extreme heat in recent years primarily affected yields by inducing moisture stress, and that the conflation of humid and dry heat extremes may lead to inaccuracy in projecting crop yield responses to warming and changing humidity. Recent research has highlighted that combinations of extreme heat and humidity are increasing in frequency with climate change3 and will continue to increase in the future4, posing particular stresses to people and other mammals5. Recent studies7 indicate that for the tropics and midlatitudes, only half of the heat accumulation in the atmosphere since the mid-20th Century is due to sensible heat , with the other half coming from latent heat (increased humidity). At the same time, heatwaves are projected to become drier (net loss of surface moisture in terms of precipitation minus evaporation) with warming in some regions9. Climate change therefore has the potential to alter the moist thermodynamic characteristics of heatwaves as circulation, precipitation, and land–atmosphere interactions respond to rising CO2.One of the known consequences of anthropogenic climate change is an increase in both regional and global mean temperatures, as well as significant increases in extreme heat event magnitudes11. While extreme heat can simultaneously cause direct thermal stress to crops13, it can also induce indirect moisture stress by raising atmospheric aridity 15. During high-temperature and high-humidity extremes, on the other hand, this indirect moisture stress is somewhat limited. Precipitation and soil moisture further complicate extreme heat impacts on crops. For instance, irrigated crop yields have been shown to be much more resistant to extreme heat than rainfed crops, indicating that yield sensitivity to heat depends on the availability of moisture17. However, wet conditions  often cool heat extremes19 and raise atmospheric humidity21. This dependence between heat and moisture raises questions of whether crop impacts are caused by heat itself, or its connection to rainfall, soil moisture, and atmospheric aridity.Crops are known to experience yield loss when exposed to extremely high temperatures23. In particular, the crop impacts of high temperature versus high-temperature and high humidity extremes, and their potentially differing connection to rainfall events, remains scarcely researched. This gap in understanding limits the ability to anticipate and prepare for yield impacts of projected increases in dry and humid heat extremes in various global breadbaskets.Understanding how heat impacts crops is essential to accurately project the risks of climate warming to global crop yields and evaluate the effectiveness of adaptation strategies. However, the extent of their impacts and the associated mechanisms remains uncertain. For example, studies disagree on the relative importance of temperature, soil moisture, and precipitation as predictors of crop yield variability8. We determine extreme dry and humid heat exposures, measured as the number of days exceeding certain dry and wet-bulb temperature thresholds , and then use multiple regression to assess the relationship between these extreme heat exposures and crop yields. Wet-bulb temperature is a metric of compound high humidity and high temperature, a strong predictor of diminished agricultural labour productivity, negative human health impacts, and decreased productivity of other large mammals such as dairy cattle5. However, the impacts of humid heat extremes on crop yields as quantified using wet-bulb temperature are yet to be elucidated. Dry and wet-bulb temperatures, which are readily available or easy to calculate from existing observational data, are direct and highly consequential measures of extreme dry and humid heat conditions. We show that while dry heat extremes strongly decrease the yields of both crops (by about double the existing estimates), humid heat extremes generally have little impact, even though their associated dry-bulb temperatures exceed reported thresholds for yield losses. We further demonstrate that humid heat extremes, but not their dry counterparts, tend to be preceded by rainy conditions. This indicates that the ultimate impact of dry heat extremes on crops depends on the co-evolution of rainfall, humidity, soil moisture, and temperature in the genesis of extreme heat conditions, with implications for projecting and adapting to future climate risk to crops.In this study, we compare the impacts of high-temperature but not high-humidity (extreme dry heat) versus high-temperature and high-humidity (extreme humid heat) on county-level corn and soybean yields in the United States. Our study covers the so-called U.S. Corn Belt  and daily maximum wet-bulb temperature (extreme humid heat) during the extended growing season  based on a 30-year climatological base period, 1981–2010. We further define dry heat extremes as days exceeding the dry-bulb temperature threshold but not the wet-bulb threshold. Both the 90th and 95th percentile thresholds are used to determine the sensitivity of our results to differing levels of extreme heat. The analysis period in this study is from 1979 to 2019, though the extreme thresholds are defined based on the 30-year climatological period.To determine whether years with frequent high dry-bulb temperature (Tmax) days coincide with years with frequent high wet-bulb temperature (Twmax) days (high temperature and high humidity), we computed the correlation between the yearly MJJAS count of extreme Tmax days and extreme Twmax days at each station. We found significant positive year-to-year correlations over regions surrounding the Great Lakes, the east coast, and the Gulf coast, between extreme Tmax and extreme Twmax days Fig. a,c. The Once the days exceeding both the Tmax and Twmax thresholds are excluded from the set of extreme Tmax days, those remaining extreme Tmax days are considered extreme dry heat days, in contrast to the extreme Twmax days (extreme humid heat days). The year-to-year correlations between the total extreme dry heat days and humid heat days Fig. b,d are sWe next consider the impact of MJJAS seasonal extreme dry and humid heat exposures during the main agricultural season on corn and soybean yields. There are significant negative correlations between these crop yields and dry heat exposure, particularly in the eastern US Figs. , S4a,c, 10. This finding suggests that the conflation of humid and dry heat extremes may lead to underestimated crop yield sensitivities to extreme dry heat. Furthermore, the negative impact is distinctly associated with dry heat exposure in regions without irrigation  higher latitudes. For corn, this narrowing is driven by less negative yield sensitivities to dry heat exposure  on crop yieldsFirst, we ask whether the differing impacts occur because extreme humid heat days are associated with lower daily maximum dry-bulb temperatures compared to the extreme dry heat days. We calculate the average Tmax across all dry heat days and that for all humid heat days, as well as their differences Fig. a–c. The 10, yet we find no widespread yield reductions due to humid heat exposure. Additionally, the Tmax range for dry heat exceeding the 90th percentile  is a key driver of crop yields, an alternate explanation for the low yield sensitivity to extreme humid heat could relate to moisture supply, rather than demand. Previous studies have shown that limiting soil moisture supply during high temperatures may be the ultimate reason for the adverse effect of heat stress on cropsWe find substantial differences in accumulated precipitation over the three days prior to extreme dry and humid heat exceedances, with more than double the amount of daily precipitation prior to humid heat days Fig. c compareExtreme dry heat is generally preceded by low daily precipitation in the days leading up to the dry heat Fig. a, and th13. However, we note that dry and humid heat impacts may further differ through a crop-climate feedback whereby stomatal closure during dry heat amplifies local canopy heating, whereas sustained transpiration during humid heat can locally buffer regional high temperatures27. This feedback may boost local crop canopy temperatures during dry heat compared to air temperatures measured at weather stations28, which may be limited during humid heat.By contrasting the crop yield sensitivities for dry and humid heat extremes, our study helps clarify the causality of heat impacts on crop yields. The absence of yield losses from exposure to humid heat days with Tmax > 30 °C suggests that extreme heat mainly reduces yields through indirect moisture stresses. This conclusion is consistent with the fact that damaging effects of dry heat extremes in this study (and in others investigating yield impacts of temperature irrespective of humidity) occur at lower temperatures (~ 30 °C) than experimental thresholds for direct thermal impacts31. Our results extend these past findings by showing how the specific combination of rainfall, humidity, and heat determines whether extreme temperatures lead to benign or damaging impacts on crops. This may help explain the previously-reported weak correlation between crop yields and seasonal mean precipitation, a metric that is agnostic of the timing of rainfall and humidity relative to extreme heat13. However, whether the lack of yield losses during humid heat extremes is attributable to plant-available soil moisture from the antecedent rainfall, as opposed to the ensuing atmospheric humidity, merits further research.The close relationship between higher rainfall directly preceding heat events and reduced crop yield loss further suggests that the timing of the rainfall relative to high heat exposure is crucial to impacts on crop yields, a novel finding of our study. Previous studies suggest that moisture supply shortages associated with dry soil conditions coupled with high temperature exposure are the key ingredients for crop yield reduction, rather than extreme heat exposure alone32. But our results here raise the possibility that irrigation effectively converts regional dry heat to local humid heat in the irrigated crop canopy, as irrigation water is readily evaporated and transpired under hot conditions, humidifying the boundary layer.The lack of strong negative impact of extreme humid heat on crop yields and the beneficial effect of rainfall immediately prior to high heat exposure begs the question of how irrigation may play a role in the relationship between dry and humid heat impact on crop yields. In Fig. 8, rainfall intensification34, and land–atmosphere coupling in a warming climate31, with complex implications for crop production. For example, positive trends in extreme dry heat incidence are already detectable for the U.S. corn growing region  and variables (e.g. vapour pressure deficit and shortwave radiation) will also be instructive. While we find that more precipitation tend to lead the humid heat extreme but not dry heat extremes, future studies could explore more fully the two-way interactions between leading precipitation and dry and humid heat via atmospheric dynamics (associated for example with moisture advection), and more local moisture recycling via the land surface (soil moisture). Since these interactions may have characteristic timescales longer than the 3 day lead and lag periods explored here, more research is needed on how yields are impacted by multivariate interactions between subseasonal precipitation and temperature at these longer timescales. Furthermore, the yield effects of relative proximity to large sources of moisture climatologically and during weather sequences surrounding heat extremes, from the Gulf of Mexico and Great Lakes especially, is worthy of further research. More research is needed on heat impacts at different stages in plant phenological cycles, and on other crops and regions. In addition, greater attention is needed to project how these relationships may change in the future and understand the likely crop consequences. Understanding the underlying mechanisms of the difference between the dry and humid heat impacts on crop yields is essential to adapt crop genetics and management to changing joint climatology of heat and moisture.27. Perhaps they sacrifice potentially-precious water—beyond what photosynthetic need and associated stomatal opening transpiration dictates—precisely to thermoregulate and avoid dangerous impacts from the higher dry bulb temperature that would otherwise exist, especially at the canopy level. These transpiration-related questions are especially interesting given the larger dry bulb temperature differences on dry heat and humid heat days in areas where crop production is less intensive. Along similar lines, it would be interesting to ask whether during rare years when the corn-belt crops truly failed catastrophically, such as during persistent dry years of the Dust Bowl period, dry bulb maximum temperatures spiked a lot higher than what we report here. Of course, other related factors besides reduced crop production and transpiration could be responsible for the larger temperature differences further west  and daily maximum wet-bulb temperature (Twmax) from 1979 to 2019 in this study. We use hourly HadISD dry-bulb temperature, and calculate the wet-bulb temperature using specific humidity, elevation, and mean sea level pressure, as in ref.3. We use the Davies-Jones method42 to calculate hourly wet-bulb temperatures, and apply the ref.43 implementation of this method using the Matlab code in ref.44. This method minimizes error at high temperatures45. Daily maximums are calculated as the maximum of hourly dry- and wet-bulb temperatures.We use the UK Met Office Hadley Centre global sub-daily station observations (HadISD) version 3.1.1.202004p45. Also, while dry-bulb temperature thresholds have been empirically established10, no such basis yet exists for wet-bulb temperatures. We apply quality control to the station data in the same way as in ref.3, in that we require data for at least 90% of the days in 90% of the months between 1979 and 2019. Stations that do not meet this threshold are removed from the analysis. The analysis here only uses the stations that are located within the county boundary that provides crop yield data  and Twmax exceed the local 90th and 95th percentile threshold (humid heat days) values based on the 30-year base period, 1981–2010. Figure https://quickstats.nass.usda.gov) county level data from 1979 to 2019. Both non-irrigated and irrigated yields are used for corn and soybeans, as indicated by the circles and triangles in Fig. The U.S. corn and soybean yield data are taken from USDA National Agricultural Statistics Service (Pearson’s correlations are used to calculate the correlation between detrended yields and extreme heat days as:0th Fig. a,c and 90th Fig. a,c perce0th Fig. b,d.DHD denotes dry heat days, HHD denotes humid heat days, t denotes years. We fit these models separately for corn and soy. The significance of the regression coefficient β1 and β2 is determined by dividing the estimated coefficient over the standard errors of this estimate and then using the two-sided t-test at the 95th confidence level.We further apply multiple regression models linking yield to dry and humid heat exposure for each county of the form:i and t denote counties and years. National-scale yield sensitivities to dry and humid heat estimated using this method are consistent with the national average of county-scale sensitivities estimated using multiple regression.As a robustness check, we also apply a national scale fixed-effect linear model on the non-detrended dataset of the form:To measure the multicollinearity between the two independent variables in Eq. ,3, the dSupplementary Figures.