Phosphorylation of the hydroxyl group of the amino acids serine and threonine is among the most prevalent post-translational modifications in mammalian cells. Phospho-serine (pSer) and -threonine (pThr) represent a central cornerstone in the cell's toolbox for adaptation to signal input. The true power for the fast modulation of the regulatory pSer/pThr sites arises from the timely attachment, binding and removal of the phosphate. The phosphorylation of serine and threonine by kinases and the binding of pSer/pThr by phosphorylation-dependent scaffold proteins is largely determined by the sequence motif surrounding the phosphorylation site (p-site). The removal of the phosphate is regulated by pSer/pThr-specific phosphatases with the two most prominent ones being PP1 and PP2A. For this family, recent advances brought forward a more complex mechanism for p-site selection. The interaction of regulatory proteins with the substrate protein constitutes a first layer for substrate recognition, but also interactions of the catalytic subunit with the amino acids in close proximity to pSer/pThr contribute to p-site selection. Here, we review the current pieces of evidence for this multi-layered, complex mechanism and hypothesize that, depending on the degree of higher structure surrounding the substrate site, recognition is more strongly influenced by regulatory subunits away from the active site for structured substrate regions, whereas the motif context is of strong relevance with p-sites in disordered regions. The latter makes these amino acid sequences crossroads for signaling and motif strength between kinases, pSer/pThr-binding proteins and phosphatases. To react and adapt to changes, cells have developed more than 400 different post-translational modifications (PTMs), which act as dynamic modulators upon attachment to amino acid (AA) side chains in order to modify protein stability, activity, structure, localization and binding . Among tAdvances in phosphoproteomics have led to a drastic increase in phosphorylation site (p-site) annotations over the last 15 years: Whereas at the beginning of the millennium the majority of the known phosphoproteome still consisted of p-sites identified and characterized by directed low-throughput (LTP) efforts aiming to study the role of single p-sites , this piAmong all p-sites, pSer is observed significantly more often than pThr and pTyr: ∼5% constitute pThr, only 0.4–4% constitute pTyr sites, with pSer being by far more abundant . When noIn this regard, many conclusions on pSer/pThr-sites were derived without considering the structural context surrounding pSer/pThr and the competing interplay between kinase, phosphatase and pSer/pThr-binding proteins. We see orthogonal insights into the regulation of the pSer/pThr phosphoproteome by the Phosphoprotein Phosphatases 1 and 2A  that regulate the majority of pSer/pThr dephosphorylation events , as a cuPhosphorylation on pSer and pThr for modulation of protein function, structure, localization and activity is not regulatory by itself, but rather needs attachment and removal in a timely and specific manner in order to exhibit its full power in cellular signaling. The biggest advantage lies in the speed by which cells can change and adapt protein function upon signal input . WhereaspSer/pThr-binding protein domains, such as WW, forkhead-associated (FHA), and 14-3-3, have also been identified to add complexity to phosphosignaling by recruiting partner proteins and serving as signaling hubs in a phosphorylation-dependent manner. For 14-3-3 proteins, the Mode 1 (RSX(pS/T)XP) and Mode 2 (RX(F/Y)X(pS)XP) consensus motifs of 14-3-3 binding sites , mostly What has not entered the equation and the more holistic view of pSer/pThr signaling, are the counteracting phosphatases. This is mainly due to the fact that the picture is less clear for the most prominent family of pSer/pThr phosphatases, namely the PPPs. This subfamily of phosphatases consists of 7 phosphatases: PP1, PP2A, PP2B , PP4, PP5, PP6 and PP7, with two of its best-studied members being Protein Phosphatase 1 and 2A , on which this review focuses on. For most members of this set of phosphatases, evolution chose a different path than for kinases. PPPs mostly consist of a highly conserved catalytic core protein. This catalytic subunit (denoted for PP1 and PP2A here as PP1c and PP2Ac) does not show high degrees of flexibility, but rather provides rigid binding interfaces for the temporary docking of a multitude of substrate-specifying regulatory subunits . These aN-terminal of the p-site for PP1 and for pThr over pSer is caused by the catalytic subunit itself [N- and C-terminal of the p-site, thereby creating specificity on the motif-level, and also the catalytic cleft creates specificity for pThr over pSer in the cases of PP1c and PP2Ac [Early findings of the 1980s and 1990s suggested that PP1c and PP2Ac exhibit an intrinsic specificity towards certain substrate motifs. These observations at the time were based either on sets of peptides synthesized in a targeted manner ,25 or ont itself . Conseqund PP2Ac ,36–38. Ind PP2Ac .In contrast to p-sites in ordered regions and Pfam domains, p-sites located within intrinsically disordered regions (IDRs) are located in sequence stretches and loop regions characterized by high flexibility that do not engage in stable secondary and tertiary conformations in their ground state. As a consequence, these IDRs are lacking structural features and binding interfaces that could potentially contribute to p-site selection for pSer/pThr-specific scaffold proteins, kinases, and phosphatases. Therefore, the p-site recognition in IDRs does not follow the classical thinking, in which secondary and tertiary protein structure determines the function of the protein region in a key/lock-like manner, also termed the function-follows-structure paradigm .Given the two-layered system for p-site recognition by PP1 and PP2A holoenzymes introduced above, we hypothesize that these layers might contribute to p-site selection with varying degrees between structured and disordered regions. However, in the current literature there is a clear bias towards studying the regulatory impact of phosphorylation sites in ordered regions  due to tTo take an exemplary look at p-sites in long (≥31 AAs) IDRs ,46 we anAlthough IDRs themselves do not exclude the possibility that secondary and tertiary structure more distant from the p-site contribute to p-site selection, the intrinsic features of IDRs give the primary amino acid sequence in direct vicinity of pSer/pThr central importance and increased relevance for determining selectivity. The importance of p-sites in IDRs has been demonstrated in very recent proteomic approaches using thermal proteome profiling and pulse-chase-based phosphoproteomic experiments: In both cases the effect of phosphorylation on protein stability was largest with p-sites located in IDRs compared with structured regions, providing evidence that a large fraction of p-sites in IDRs serves a regulatory function ,52. AlsoWhile amino acid composition alone is not sufficient to identify and classify IDRs , work byThere is solid evidence for the importance of IDRs in interacting with the rigid catalytic core of PP1 and PP2A: For PP1, it has been demonstrated that more than 70% of its regulatory proteins constitute intrinsically disordered proteins (IDPs). For example, the complex of PP1 with its myosin-targeting subunit MYPT1 (PP1:MYPT1) , spinophN-terminus of Histone 3 (H3) [It is therefore an intriguing idea to extend this paradigm to the regions on substrate proteins dephosphorylated by PP1c and PP2Ac: Since many of the PP1c and PP2Ac sensitive p-sites are located in IDRs lacking structural features for surface recognition, the primary amino acid context surrounding pSer/pThr gains special importance for interacting with the rigid grooves surrounding the catalytic cleft of these phosphatases. This places a layer of PPP specificity at the same level as motifs of kinase and pSer/pThr-binding proteins and makes these amino acid sequences crossroads for signaling and motif strength between enzyme classes. In fact, several cases are known for such a scenario: PP1/Repo-Man regulates the highly disordered e 3 (H3) ,70 by dee 3 (H3)  and S10 e 3 (H3) ,72 to ree 3 (H3) . Similare 3 (H3) , which ie 3 (H3) . Also, de 3 (H3) , again ae 3 (H3) . Indeed,Altogether, the following model of PP1/PP2A substrate specification has evolved: As a first layer for substrate targeting, PP1 and PP2A associate with regulatory subunits. This step can also lead to binding of a regulatory subunit close the catalytic subunits’ surface surrounding the catalytic cleft, which in turn alters the motif preference of the holoenyzme. The second layer of p-site selectivity is determined by the catalytic subunit and its surface properties as such and is actively contributing to the overall specificity of the holoenzyme . These lThe success in interpreting the exact role of more than 100 000 human p-sites in health and disease is largely dependent on deciphering a complex system between kinases, p-motif binding proteins and the multilayer specificity of phosphatases in the light of motif context and structural information. Given the recent insights presented in this review, we anticipate that disentangling the phosphoproteome in terms of sequence context and structural features will strongly help to elucidate the regulatory relevance of p-sites and the mechanism of p-site recognition by PP1 and PP2A in the upcoming years, and will support completing the understanding of the interplay between kinases, phosphatases, and interacting proteins with phosphoprotein p-sites.Whilst data for the human phosphoproteome currently comprises more than 100 000 identified phosphorylation sites, only a small fraction has been studied in terms of regulation by kinases, phosphorylation-dependent binding proteins, and phosphatases.The importance for the sequence context of substrate sites for Ser/Thr kinases and pSer/pThr-binding proteins is quite well established, however, a more complex model for the pSer/pThr phosphatases PP1 and PP2A is currently evolving.For these phosphatases, further studies are needed to differentiate between substrate recognition by regulatory subunits distant from the pSer/pThr substrate site and motif recognition in direct vicinity of the pSer/pThr by the catalytic subunit. We hypothesize that these layers affect substrate recognition differently in structured and disordered protein regions.


Influenza B viruses (IBV) circulate annually, with young children, the elderly and immunocompromised individuals being at high risk. Yearly vaccinations are recommended to protect against seasonally influenza viruses, including IBV. Live attenuated influenza vaccines (LAIV) provide the unique opportunity for direct exposure to the antigenically variable surface glycoproteins as well as the more conserved internal components. Ideally, LAIV Master Donor Viruses (MDV) should accurately reflect seasonal influenza strains. Unfortunately, the continuous evolution of IBV have led to significant changes in conserved epitopes compared to the IBV MDV based on B/Ann Arbor/1/1966 strain. Here, we propose a recent influenza B/Brisbane/60/2008 as an efficacious MDV alternative, as its internal viral proteins more accurately reflect those of circulating IBV strains. We introduced the mutations responsible for the temperature sensitive (ts), cold adapted (ca) and attenuated (att) phenotype of B/Ann Arbor/1/1966 MDV LAIV into B/Brisbane/60/2008 to generate a new MDV LAIV. In vitro and in vivo analysis demonstrated that the mutations responsible of the ts, ca, and att phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to infer the same phenotype to B/Brisbane/60/2008, demonstrating its potential as a new MDV for the development of LAIV to protect against contemporary IBV strains. Over the last century, influenza viruses have been responsible of both yearly seasonal and occasional pandemics infections of great consequences to humans. While Influenza A viruses (IAV) circulate within several zoonotic hosts, providing the opportunity for pandemic, Influenza B viruses (IBV) primarily circulate in humans, and are not considered a pandemic threat. However, IBV are still major human pathogens responsible for seasonal epidemics of respiratory illness ,2,3. IBVOrthomyxoviridae family /mL). Briefly, confluent monolayers of MDCK cells  were infected with 50 μL of virus from each time point (in triplicate) using 10-fold serial dilutions. Plates were placed at 33 °C for 12 h. After viral infection, cells were fixed and permeabilized with 4% formaldehyde and 0.5% Triton-X100 in 1× PBS. A primary monoclonal antibody against IBV NP  was added to each well and incubated at 37 °C for 90 min. After 3× washes with 1× PBS, a secondary FITC-conjugated polyclonal rabbit anti-mouse antibody (Abcam Cat. ab6724) was added to each well and incubated at 37 °C for 45 min. After 3× washes with 1× PBS, plates were observed under an Olympus fluorescent microscope to quantify positive stained cells.Confluent monolayers of MDCK cells  were infected with B/Brisbane/60/2008 WT or MDV LAIV for 1 h at room temperature. After 1 h infection, cells were overlaid with DMEM/F-12 containing 2% Oxoid agar and incubated at 33 °C or 37 °C in 5% CO2. At 72 h post-infection, cells were fixed overnight with 2.5% formalin (in 1× PBS) at 4 °C. After overnight incubaton, agar overlays were removed and cells were fixed and permeabilized using 4% formaldehyde with 0.01% Triton-X100 in 1× PBS at room temperature for 1 h. Immunostaining of viral plaques was performed using the IBV anti-NP monoclonal antibody B017 and vector kits (Vectastain ABC kit and DAB HRP Substrate Kit: Vector), according to manufacturer’s specifications.Confluent monolayers of MDCK cells  was anesthetized as described above and treated with sterile 1× PBS as a negative control [n = 3) and 4 (n = 3) post-infection [6 FFU). Mice were euthanized at day 2 and 4 post-challenge. Lungs were harvested and viral titers determined as described above [Six-week-old, female C57BL/6J mice were obtained from the National Cancer Institute (NCI) and maintain in the University of Rochester Animal Care Facilities under protocols outlined by the University Committee of Animal Reseources (UCAR). Mice were anesthetized through intraperitoneal injection of Avertin (240 mg/kg of body weight) and subsequently infected intranasally with 10MDV LAIV . An addi control . To detenfection . Harvestnfection . Serum fnfection . On day ed above .2SO4 and read at 450 nm using the Vmax kinetic microplate reader (Molecular Devices).To determine total IBV specific antibodies, serum collected at day 14 post-infection from infected mice was used in ELISA assays. Briefly, 96-well plates were coated at 250 ng/well with either B/Ohio/01/2005  or B/Malaysia/2506/2004  recombinant HA proteins and placed at 4 °C overnight. Plates were then blocked with 3% BSA in 1× PBS for 1 h at 37 °C. Serum diluted in 0.5% BSA in 1× PBS was then added to the plate using two-fold dilutions, starting 1:100 dilution, and incubated at room temperature for 2 h. Plates were washed 3× with ELISA wash buffer (1× PBS + 0.05% Tween) and then 50 μL of a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody  was added to each well for 1 h at 37 °C. Reactions were developed with tetramethylbenzidine (TMB) substrate (BioLegend) for 10 min at room temperature. Reaction was stopped with 2N HSerum from mice infected with either B/Brisbane/60/2008 WT or MDV LAIV at day 14 post-infection were RDE (receptor-destroying enzyme) treated overnight at 37 °C and then placed at 56 °C for 30 min for heat inactivation. To determine the presence of neutralizing antibodies, heat inactivated serum were diluted in 96-well V-bottom plates and incubated with 4 hemagglutinating units (HAU) of B/Brisbane/60/2008 WT and left at room temperature for 60 min. Then, 0.5% turkey red blood cells were added to each well and left on ice for 45 min. HAI antibody titers were calculated based on the last well in which hemagglutination did not occur, with the riciprocal of the corresponding dilution being the hemagglutination titer.To evaluate neutralizing antibodies against B/Brisbane/60/2008 WT elicited by infection with either WT or MDV LAIV B/Brisbane/60/2008, serum from infected mice were collected at day 14 post-infection, treated as outlined above, and serially diluted in 96-well plates. B/Brisbane/60/2008 expressing mCherry (100 FFU/well) ,75 was aThe first step towards identifying a new IBV MDV LAIV was to identify the ideal viral candidate. B/Brisbane/60/2008 was selected as the potential MDV LAIV candidate because this Victoria lineage IBV circulated more recently (2008) within the human population and because its internal genes have increased percent identity and sequence homology with contemporary IBV strains circulating in humans, relatively to the current B/Ann Arbor/1/1966 MDV LAIV  and lower (lungs) respiratory track were evaluated at days 2 and 4 post-infection  why LAIV had low effectiveness since the 2013-2014 season remains unknown. One of the potential explanations for the lack of effectiveness of LAIV is the use of same IAV and IBV MDV backbones. If this is the case, updating the MDV of IAV (A/Ann Arbor/6/60 H2N2) and IBV (B/Ann Arbor/1/1966) to different and more contemporary strains backbones, could solve this problem and provide with more effective LAIV for the treatment of both IAV and IBV infections. We have previously shown how the ts, ca, and att mutations of the US MDV A/Ann Arbor/6/60 H2N2 were able to confer a similar phenotype to other IAV strains, including PR8 ,62 and p


Observation of small ruminants showing differing degrees of leanness or emaciation is a common occurrence at slaughterhouses. Although either condition may result from similar causes, emaciation and cachexia are usually pathological conditions, warranting total condemnation of the carcass during post-mortem inspection. This is done in order to prevent unfit meat from entering the human food chain. Scrapie, a naturally occurring transmissible spongiform encephalopathy in small ruminants, is characterized by loss of body conditioning and a wasting appearance. Since atypical scrapie has been identified as occurring at a low, but very consistent, the prevalence in small ruminant populations, it is advisable to include this disease as a differential diagnosis of wasting conditions detected during meat inspection at the abattoir. Vigilance for detection of putative scrapie in slaughterhouses, and its differential diagnosis from other conditions associated with wasting carcasses, are of paramount importance for post-mortem decisions on the fate of carcasses, offal, as well as animal by-products.Wasting disease in small ruminants is frequently detected at slaughterhouses. The wasting disorder is manifested by the deterioration of the nutritional and physiological state of the animal indicated by thinness, emaciation, and cachexia. Evidence of emaciation and cachexia, alone, are pathological conditions leading to carcass condemnation during an inspection. Several diseases are associated with a wasting condition, including scrapie, pseudotuberculosis, tuberculosis, paratuberculosis, Maedi Visna, and tumor diseases. On the other hand, parasitic diseases, nutrition disorders, exposure or ingestion of toxins, metabolic conditions, inadequate nutrition due to poor teeth, or poor alimentary diet are conditions contributing to poor body condition. Classical and atypical scrapie is naturally occurring transmissible spongiform encephalopathies in small ruminants. The etiological agent for each one is prions. However, each of these scrapie types is epidemiologically, pathologically, and biochemically different. Though atypical scrapie occurs at low incidence, it is consistently prevalent in the small ruminant population. Hence, it is advisable to include differential diagnosis of this disease, from other possibilities, as a cause of wasting conditions detected during meat inspection at the abattoir. This manuscript is a review of the measures in force at the abattoir for scrapie control, focusing on the differential diagnosis of gross lesions related to wasting conditions detected in small ruminants during meat inspection. Antemortem and post-mortem inspection of animals is an important process for detecting zoonotic diseases and is a vital component of the One Health concept. Moreover, antemortem and post-mortem inspection are also inherently important sentinel tools for the detection of animal diseases, in general, requiring additional diagnostic tests for suspicious cases.Due to the relatively low economic value of small ruminants, post-mortem examination is an inexpensive and commonly used tool for monitoring diseases or other conditions. Post-mortem inspection is the most useful tool for achieving a pathological diagnosis and, in many cases, the only way to identify the majority of lesions on these animals. To avoid overlooking gross pathological findings, the post-mortem inspection must be performed methodically, always following the same approved and certified procedures. Visual inspection can be complemented with additional diagnostic tools. The European Food Safety Authority (EFSA) regarding human health hazards during inspection of meat from sheep and goats recommends omitting palpation and incisions to the greatest extent possible. This recommendation renders the post-mortem inspection as being essentially a visual procedure. However, the official veterinarian practice dictates additional post-mortem inspection procedures in certain instances. These additional procedures require incision and palpation of carcasses and offal when there is any indication of a possible risk to human or animal health and welfare . PresentThe wasting condition can be commonly observed in small ruminants during meat inspection. The wasting condition can result from poor nutrition, emaciation, or cachexia. Although emaciation and cachexia can result from similar etiologies, they are pathological conditions resulting in carcass condemnation during meat inspection. In animals designated as thin, there is a marked reduction of fat, but, otherwise, the animal has a normal appearance and texture . EmaciatSeveral conditions and diseases can be related to emaciation, including scrapie . This disease presents as clinical signs of changes in typical animal behavior, chiefly incoordination of gait and trembling. Classical and atypical scrapie in small ruminants is naturally occurring transmissible spongiform encephalopathies (TSEs) caused by prions. The two TSEs are epidemiologically, pathologically, and biochemically different. Since atypical scrapie has a low, but consistent, the incidence in small ruminant populations, it is advisable to include this disease for differential diagnosis of wasting carcasses detected during post-mortem inspection. Generally, this condition is linked with chronic wasting diseases such as Maedi-Visna, paratuberculosis, caseous lymphadenitis, tuberculosis, and severe cases of parasitic diseases, tumor diseases, toxic or metabolic conditions, or inadequate nutrition due to poor teeth or poor alimentary diet.This manuscript reviews clinical signs and indicator lesions to differentiate the causes of wasting conditions in carcasses of small ruminants at the slaughterhouse. The differential diagnosis in the slaughterhouse of gross lesions associated with the wasting condition is fundamental to food safety, including determination of treatment and destination of animal by-products. The main focus of this paper is to emphasize differential diagnosis of putative scrapie relative to other wasting conditions/diseases. The review also covers recommended certified laboratory tests and measures at the abattoir for scrapie control.Sc) of a normal host glycoprotein (cellular prion protein PrPc) encoded by the PrP gene (prnp). The first reported case of CS occurred in the United Kingdom in 1732. This disease was transmitted further through the export of live animals to western European countries, eventually reaching a worldwide distribution, except for Australia and New Zealand [Classical scrapie (CS), the ancient animal transmissible spongiform encephalopathy affecting sheep and goats, is a naturally transmitted infectious and fatal neurodegenerative disease, reportable to the World Organization for Animal Health (OIE). The disease state results from the accumulation of a misfolded form . This is followed by a long asymptomatic incubation period, usually ranging between two and seven years, during which infected animals are a source of further contamination. The clinical phase of the disease can last several months. Initially, the disease is characterized by a progressive loss of body weight (with preserved appetite) and slight behavioral changes, occurring for short periods that may go unnoticed. Later stages of the disease present progressive pruritus accompanied by hyperesthesia and uncoordinated gait .Sc within affected sheep, as well as genotype of animal affected and associated epidemiology  was identified in Norway . The twoemiology . AtypicaEpidemiological studies have failed to confirm exposure to small ruminants, or their products is a risk factor for zoonotic spill-over of prionic diseases to humans. This finding has led to the conclusion small-ruminant prion-diseases present a negligible risk of developing into a zoonotic outbreak. However, recent findings show the prevalence and geographical distribution of TSEs in small ruminants have been underestimated, in addition to the observation of transmission of CS in non-human primates  and humaScrapie can be caused by several strains of prions, but the diversity of these strains in small ruminants remains undefined. At least five different ovine prionic strains are known, including AS . These sAll Member States (MS) of the European Union (EU) developed and implemented scrapie surveillance, control, and eradication programs, respective to each EU country. These include passive surveillance, involving the investigation of animals with neurological signs, and active surveillance, entailing sampling of small ruminant populations from specific groups of animals based on fallen stock, those for emergency slaughter, animals with clinical symptoms of diseases other than TSEs during the antemortem inspection, and healthy animals slaughtered for human consumption . The eraCS is still the most frequently reported type of prionic disease in the EU in goats and sheep, but with a differing incidence between member states. Over the past 10 years (2010 through 2019), the incidence per 10,000 tested animals, considering both CS and AS and both goats and sheep, showed a range between 0.5 and 5.4. However, there has been a statistically significant decreasing trend for both CS and AS in sheep, but not yet verified in goats . InternaAn essential component for the control of TSEs in slaughterhouses also includes correct classification and consequent processing of animal by-products. Concerning the zoonotic potential of classical BSE, the EFSA amended prior requirements for the removal of specified risk material (SRM) in small ruminants in the slaughterhouse. The amended procedure recommends those tissues having the highest levels of BSE infection in small ruminants be designated as SRM according to their classification in Category 1 by-products . In thisAccording to Regulation (EC) No 1069/2009 , the entThe antemortem clinical presentation of the two types of scrapie can include weight loss or emaciation, behavioral changes , trembling, and uncoordinated gait. Pruritus may also be present in CS, as opposed to AS. As these clinical signs may be considered as suggestive for scrapie, at the abattoir, varying combinations of these signs and degree of severity can be observed. In some circumstances, a combination of these clinical signs may not be scrapie-specific, leading to misdiagnosis. For example, staggering, self-isolation, and trembling are signs that can also be present in other wasting diseases, most notably in a weak condition. In these cases, other antemortem signs  and post-mortem findings  could be of significant importance for directing a diagnosis, in view of emaciation in the absence of other lesions, on post-mortem inspection, is an indication of scrapie. Antemortem inspection is highly important for detecting and registering any neurological disease that may not present any observable macroscopic causation during post-mortem inspection. Such cases could be scrapie. But, in some circumstances, when clinical signs are not obvious or antemortem inspection  does not permit detection of neurological abnormalities, suspected cases of scrapie must be based on detection of wasting carcasses.Post-mortem inspection may only present emaciation of carcasses as the only finding of either form of scrapie. But, as aforementioned, wasting conditions can result from a multiplicity of factors, namely infectious diseases, or deficient animal care . Thus, pathological observations on post-mortem inspection may be a decisive factor for differential diagnosis.A graphical categorization of the main wasting diseases/conditions, other than scrapie, seen during meat inspection is presented in Selection of diseases/conditions associated with wasting conditions was established through thorough discussions with a group of multidisciplinary stakeholders , based on the totality of condemnation data at the abattoir and respective anatomopathological and microbiological findings, in Portugal.Several infectious diseases can lead to a chronic wasting syndrome, characterized by progressive weight loss, including the following: caseous lymphadenitis (CLA), pseudotuberculosis ,29,30, tThe main antemortem signs and gross post-mortem lesions observed in animals/carcasses with corresponding chronic wasting from infectious  diseases are listed in The presence of malignant tumors in sheep and goats generally is associated with the declining condition. At the time they reach the slaughterhouse, animals exhibiting such tumors are generally relatively old , having aged long enough to develop gross tumoural lesions . There aThe majority of observed malignant tumors are spontaneous and their etiology has not been recognized so far, with the exception of two neoplasms, nasal carcinomas of sheep and goats, and ovine pulmonary adenocarcinoma (OPA). Pulmonary adenocarcinoma is commonly recorded as an incidental finding in the abattoir ,62. SheeClinical signs and gross lesions observed in emaciated small ruminants resulting from the presence of malignant tumors are outlined in Subclinical and chronic parasitic infections are considered major causes of economic loss in small ruminants exhibiting poor physical condition and chronic wasting at pasture ,67. SympNutritional disorders can also be associated with wasting carcasses of small ruminants ,79. In mPrimary nutritional conditions (disorders) leading to poor body condition may result from inadequate energy and protein levels or a lack of specific macro and microminerals. These inadequacies may be due to poisonous pasture plants, leading to direct toxicity of the animal, or deficiencies in soil nutrients for, or diseases in, forage plants, leading to repercussions in maintaining normal ruminal flora ,78. CrudDiagnosis of wasting as a result of nutritional deficiencies is not easily differentiated from scrapie at antemortem examination even if neurological abnormalities associated with scrapie are observed. Moreover, at post-mortem inspection of nutritional diseases, as with scrapie, other injuries or anomalies may not be easily detected. Neurological signs of scrapie in infected animals may not always occur, particularly in atypical scrapie. In such cases, laboratory diagnosis is essential.Secondary nutritional disorders may be related to an inability to obtain or absorb nutrients from available food. Such deficiency may lead to a variety of problems, including locomotor problems , blindness , diseases of the oral cavity  and oral cavity anomalies . With secondary nutritional disorders, observation of antemortem signs is often associated with post-mortem lesions that serve as the basis for differential diagnosis of these nutritional disorders from scrapie.Conditions for differential diagnosis of wasting condition at the slaughterhouse, based on antemortem signs and post-mortem gross lesions, are summarized in Sc in these tissues, using an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, or western immunoblots. Histopathological methods may lead to detection of vacuolar lesions in the CNS [There is no validated laboratory test for the diagnosis of scrapie in living animals. Definitive diagnosis of scrapie currently requires post-mortem laboratory tests using tissue samples of the central nervous system (CNS) in authorized laboratories and/or in the national reference laboratory for animal TSEs. These tests are designed to detect the presence of PrP the CNS  having pSc approved by the European Commission, listed in Annex X of Regulation (EC) No. 999/2001 [There are a number of commercially available rapid test kits for detection of PrP999/2001  as last 999/2001 .The judgment (sanitary decision) if meat is fit for human consumption, has to be based on two fundamental aspects: (1) degree of deterioration of the carcass, and (2) cause of loss of body condition. A correct diagnosis of the causes of the wasting condition  is essential, since it has major consequences to the sanitary processing of the carcass, as implemented by the official veterinarian, as well as in the classification of the carcass for determining the subsequent destination of animal by-products.Judgment is based on the degree of loss of condition, presence of concurrent diseases, and eventually results of laboratory examinations. Particular attention has to be given to extent of emaciation, presence/absence of edema, and current diseases. This condition, especially if not associated with concurrent diseases, is among the most difficult to assess during meat inspection.This is particularly the case depending on the locality where official conditional approval for manufactured purposes and/or heat treatment is not authorized. Note that EU Meat Inspection Regulation , only predicts conditional approval to “meat not generally infected with cysticercus in which parts uninfected may be declared fit for human consumption after having undergone a cold treatment”. This is particularly the case in regions where conditional approval for manufactured purposes and/or heat treatment is not authorized. Note that EU Meat Inspection Regulation  , even after a concurrent disease and post-mortem inspection reveal localized lesions of one organ or part of the carcass, only the affected organ or portion of the carcass, and associated lymph nodes, shall be declared unfit for human consumption. Thinness may be physiologically normal  as occurAnimal products from animals not fit for consumption could be a potential source of risk to public and livestock health. Regulation (EC) N° 1069/2009  introducThe considerable seriousness in the detection of scrapie to public and livestock health, and its potential for severe economic loss, demands the participation of an Official Veterinarian at the abattoir in order to guarantee careful and detailed inspection procedures and issuance of sanitary decisions. The judgment made for the fate of carcasses and any by-products requires an assessment of information compiled during ante- and post-mortem inspections, under the purview of veterinarian expertise and analytical reasoning.Many different situations may account for wasting detected in animal/carcasses at the abattoir. Some of these conditions are devoid of public/animal health concerns. However, among these diverse situations leading to a poor carcass condition, some may involve animal welfare (poor care), infectious diseases, or zoonoses, such as TSEs. Moreover, in some of these cases, a definitive diagnosis is not available by macroscopical pathological examination. In such cases, laboratory procedures may be required. The laboratory diagnosis of a scrapie suspicion is mandatory, as it does not cause macroscopic lesions to be observed on post-mortem inspection. Failure to use laboratory tests to diagnose scrapie would result in the misevaluation of the carcass, and consequent incorrect classification of by-products. Missing the diagnosis of scrapie can have serious repercussions in terms of animal health and zoonotic spill-over to humans.


In India, the construction sector provides the main alternative to agricultural work - seasonal migration to and from construction work is widespread and construction work remains the second-largest employer of women in the country behind agriculture. Occupational injuries, which kill over 300,000 people annually, are a serious public health concern. However, data on construction site injuries to women are lacking, as India does not publish statistics on occupational injuries and illnesses. Our objectives were to: Estimate the number of women injured in construction site accidents in Delhi; and to estimate and compare the annual construction site injury rates per 100,000 workers of males and females in Delhi.We conducted a two-sample capture-recapture study using data for accidents reported to the Delhi Police, Employee State Insurance Corporation (ESIC), and Commissioners of Workmen Compensation (CWC) of Delhi Government. The capture-recapture method has been used in epidemiology, to estimate morbidity and mortality using multiple, overlapping, but incomplete data sources. This study is based on the injuries reported from construction site accidents in Delhi in 2017. We linked the data from each of the data sources using the name, gender, and age of each injured person, the date and place of the accident, and the name of the employer. We used the Chapman estimator to estimate the total incidence of construction injuries in Delhi.p = 0.001) that the overall construction site injury rate per 100,000 workers of females was about one half the rate of males [rate ratio 0.56 (95%CI: 0.40 to 0.78)]. There was no evidence (p = 0.601) that the rates of fatal injuries differed in males and females (rate ratio 1.14 (95%CI: 0.70 to 1.87).We estimated that there was a total of 37 female construction site workers injured  in Delhi in 2017. There was a total of 1043 male construction site workers injured . FIRs ascertained two-thirds (68%) of all injuries to females but only one third (34%) of those to males. The annual construction site injury rate per 100,000 workers of females was 82.26 (95%CI: 57.92 to 113.39). The annual construction site injury rate per 100,000 workers of males was 146.5 (95%CI: 137.7 to 155.6). There was strong evidence  that are appropriate and culturally acceptable to Indian women are needed. In India, the construction sector provides the main alternative to agricultural work - seasonal migration to and from construction work is widespread and construction work remains the second-largest employer of women in the country behind agriculture . Women aOccupational injuries, which kill approximately 335,000 persons annually, are a serious public health concern , 5. HoweDelhi, the capital of India entices construction workers due to high wages and geographical advantage . PreviouHealth records are a potential source of data on injuries across the globe. However, in India health records are generally maintained in paper format only. Even where hospital functioning has been computerised, records are often stored in disparate computer systems without inter-operability or cross-sharing . The repInformation relating to an accident, whether received orally or in writing, is required under the Indian Penal Code 1860 to be recorded by the officer in-charge of a police station, in a prescribed format, commonly known as the ‘First Information Report’ (FIR) . Informato estimate the number of women injured in construction site accidents in Delhi;to estimate and compare the annual construction site injury rates per 100,000 workers of males and females;to estimate the completeness of ascertainment by FIRs of injured female construction site workers.In this study we investigate the incidence of construction site injuries to women in Delhi. Specifically, we aimed:This was a two-sample capture-recapture study. The Capture-recapture method has been used in epidemiology, to estimate morbidity and mortality using multiple, overlapping, but incomplete data sources . The metBuilding or other construction work was defined as the construction, alteration, repairs, maintenance or demolition of buildings, streets, roads, railways, tramways, airfields, irrigation, drainage, embankment and navigation works; flood control works; generation, transmission and distribution of power; water works; oil and gas installations; electric lines; wireless, radio, television, telephone, telegraph and overseas communications; dams, canals, reservoirs, watercourses; tunnels, bridges, viaducts, aqueducts, pipelines; towers, cooling towers, and transmission towers .Building or construction worker was defined as a person who is employed to do any skilled, semi-skilled or unskilled work which could be manual, supervisory, technical or clerical in connection with any building or other construction . The defConstruction site was defined as a place where any building or other construction work is occurring .Accident was defined as “an unintentional event characterized by the sudden release of an external force or impact, which can manifest itself as body injury” .Injury was defined as “a bodily lesion resulting from acute overexposure to energy, which interacts with the body in amounts or rates that exceed the threshold of physiological tolerance” . .InjuriWe obtained the data for accidents reported to the police, Employee State Insurance Corporation (ESIC), and Commissioners of Workmen Compensation (CWC) of Delhi Government from 1st January 2017 to 31st December 2018. The study was restricted to accidents occurring between 1st January 2017 to 31st December, 2017. Accidents that happened during this period but were reported in the year 2018 to any of the three authorities were included. The accidents which happened before 1st January 2017 but reported during the year 2017 were excluded from the study. FIRs of construction site accidents were downloaded from the Delhi Police website and data were extracted . The fatal construction site injury rate per 100,000 workers of females was 37.8 (95%CI: 22.02 to 60.52) The fatal construction site injury rate per 100,000 workers of males was 33.15 (95%CI: 29.05 to 37.66). There was no evidence (p = 0.601) that the rates of fatal injuries differed in males and females (rate ratio 1.14 (95%CI: 0.70 to 1.87).The annual construction site injury rate per 100,000 workers of females was 82.26 (95%CI: 57.92 to 113.39). The annual construction site injury rate per 100,000 workers of males was 146.5 (95%CI: 137.7 to 155.6). There was strong evidence  that are appropriate and culturally acceptable to Indian women are needed. However, given the susceptibility of women workers to be laid off or under-employed, it is possible that training and safety equipment requirements might provide a further obstacle to their employment within construction.It is possible that some female workers are at increased injury risk as they are required to attend to their children playing at the construction site . The BuiThe differences in ascertainment by FIRs of fatal and non-fatal injuries according to gender could be due to under-reporting to the ESIC and the CWC of non-fatal injuries sustained by female construction workers which could have biased our estimate of the total numbers of injuries: In capture-recapture analysis, if one of the sources of data captures very few cases, the analysis is likely to produce a biased estimate of the population size . Since tThe study also points towards substantial underreporting of injuries to various authorities despite the legal mandate. The underreporting was higher for the Employee State Insurance Corporation and Commissioners of Workmen Compensation. This implies that the construction workers are being denied the entitled compensation, and insurance benefits. To prevent information from reaching enforcement agencies, the employers also try to get the workers treated at construction sites, compromising on the quality of care, which may have serious health consequences. It also informs that the employers are not being punished for various acts of their negligence which are leading to accidents and injuries at the construction sites. The results of this study also point towards an immediate need to create awareness about the legal provisions regarding reporting of injuries and stricter measures to enforce the laws. The construction industry in India is under-supervised due to country wide shortage of Labour Inspectors and other enforcement staff.The study also underscores the need to have a comprehensive injury prevention policy in India. It highlights the need for creation of a unified authority for injury prevention and mitigation under the Ministry of Health and Family Welfare in India, a responsibility scattered across multiple departments.Our research was confined to Delhi, the capital of India. For a national picture of the burden of injuries to female construction workers, a national study is needed. Findings of such a study may stimulate appropriate policy responses at the national level as well as in different states of India. Estimates of the injury burden and gender differences may be validated in future studies using other sources of data on injuries for the ‘recapture’ sample . Future research might also focus on the burden of other occupational injuries  using a similar research design.


DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.The specificity of inhibition by 6,6′-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC Nuphar lutea (L.) Sm. (Nymphaeaceae) contains a unique family of bioactive sesquiterpene thioalkaloids, nupharidines -AVLQSGFR-[Lys(Dabcyl)]-K-amide). Mpro was pre-incubated for 15 min at room temperature with compound before the addition of substrate. Protease reaction was measured continuously in a BMG Pherastar FS with a 480/520 ex/em filter set. Data were mapped and normalized in the Genedata Screener.The assay was conducted according to . CompounNuphar lutea (L.) Sm. (Nymphaeaceae) was investigated as an inhibitor of Cathepsins and other proteases for comparative purposes. DTBN shows differences in the extent of Cathepsins inhibition, reflecting structural-steric and biochemical differences in the active site. Cathepsin S was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain. DTBN was inactive for the inhibition of SARS-CoV-2 Mpro of COVID-19 and non-cysteine proteases, Trypsin and Calpain. Docking simulations suggested a mechanism of interaction that was supported by the biochemical result. In the docking results, it was shown that the active site cysteine sulphur of Cathepsin S, L and B is in close proximity to the nupharidine tetrahydrothiophene ring, potentially creating the necessary conditions for a nucleophilic attack. Covalent docking of intermediate DTBN (iDTBN) to the active site of Cathepsins led to a disulfide bond between the electrophilic sulphur of iDTBN and nucleophilic cysteine sulphur of Cathepsins. This explains the potential of electrophilic sulphur in DTBN as active warhead that inhibits nucleophilic cysteine sulphur containing enzymes. Further, molecular dynamic (MD) simulation revealed the stability of protein–ligand complexes, where Cathepsin S formed the more stable complex with DTBN and iDTBN in comparison with Cathespsins B and L. The order of stability was in accordance with in vitro results, emphasizing the importance of the stability of the protein–ligand complex for inhibition. DTBN’s lack of reactivity against SARS-CoV-2 Mpro was attributed to a mismatch of binding conformation of DTBN to the catalytic binding site of SARS-CoV-2 Mpro. Our results may be conducive for a mechanism-based search of derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.Cathepsins, a family of proteases expressed in all living organisms, including humans, participate in protein degradation, cell signaling and protein-processing. Abnormal expression and/or activity of Cathepsins has been associated with a variety of human diseases, including cancer, inflammatory and cardiovascular diseases, neurodegenerative disorders, viral diseases, and many others. 6,6′-Dihydroxythiobinupharidine (DTBN), a bioactive sesquiterpene thioalkaloid, is present in yellow water lily,


Pedicle screw fixation has been the gold standard because this technique allows solid arthrodesis and provides a degree of stiffness that immediately diminishes mechanical back pain. Although the translaminar screw technique is considered inferior to the pedicle screw technique due to the thought that it is minimally invasive, studies proved that both fixation systems had significantly greater stiffness and reduced range of motion compared with the normal vertebrae. The purpose of this study is to determine the fusion rate, the clinical outcome of translaminar screw fixation of the lumbar and lumbosacral for the long term.We evaluate six patients with a degenerative lumbar disorder and performed posterior lumbar fixation and fusion with the translaminar screw. The translaminar screw was performed monosegmentally (3 patients), across two segments (1 patient), and across three segments (2 patients). We then evaluate Oswestry Disability Index (ODI) score one week, three months, and one year postoperatively.When lumbosacral spine fusion procedures are performed without supplemental internal fixation, a 10% pseudoarthrosis rate can be expected for single-level fusions, and the percentage can reach 30% if more than two levels are fused. Compared to the pedicle screw technique, the translaminar technique provides a limited profile and less bony invasion that will minimize the risk of failure.Translaminar screw offers immediate postoperative stability, seen in postoperatively, patients experienced better quality of life than preoperatively. It also represents a useful and inexpensive technique for short segment fusion of the non-traumatic lumbar and lumbosacral spine. •Subjective assessment showed all patients experienced symptoms relieved post-surgery.•The translaminar technique provides less bony invasion to minimize the risk of failure.•The biomechanical test reveals translaminar screw increased the stiffness 2.4 times.•This technique can be an alternative for fusions in the degenerative lumbar disease. However, its safety, insertion facility, and morbidity have been clearly and consistently reported in the literature. In addition, this technique also jeopardizes the paraspinal muscle due to traction and has been shown to injure these muscles functionally and increase the volume of devitalized tissue, leading to an increased incidence of infection with additional complications such as dural tears, neurological injury, and an increased rate of infection due to long operating time Although the translaminar screw technique is considered inferior to the pedicle screw technique due to the thought that it is minimally invasive, one study conducted by Ferrara et al. proved that the translaminar screw technique was found to be equivalent to bilateral pedicle screw placement The purpose of this study is to determine the fusion rate, the clinical outcome of translaminar screw fixation of the lumbar and lumbosacral for the long term. The author report six cases of posterior instrumentation using translaminar screw technique followed up with subjective assessment using ODI (Oswestry Disability Score) preoperatively and postoperatively, and objective assessment using radiographic evaluation 2We present six cases of degenerative lumbar disorder with a history of back pain more than one year that underwent posterior stabilization using the translaminar screw technique described by Magerl , Fig. 5.The radiologic investigation was done by anteroposterior, flexion-extension, and lateral x-ray film to detect segmental motion, 12 months postoperative. A solid bony fusion with a radiologically calcified fusion mass, no apparent motion on the flexion-extension x-rays in the fused segment was documented in all patients, there were no Perioperative or late infections, and there was no neurologic injury observed .Fig. 2A Loosening of the screw (radiolucent area) was noted in one patient (fixation across three segments). However, no segmental motion could be detected on the flexion-extension radiographs. The screw fragments remained without causing any symptoms, and no migration was detected. The radiolucent halo surrounding the implant was found in one patient. This line was measured, and the length was more than 1 mm, which was considered positive for osteolysis. This sign indicates instability on the site of arthrodesis . HoweverEach patient completed a questionnaire to evaluate subjective results using the ODI score . Based o3When lumbosacral spine fusion procedures are performed without supplemental internal fixation, a pseudoarthrosis rate of about 10% can be expected for single-level fusions, and the percentage can reach 30% if more than two or three levels are fused Five fundamental requirements will influence fusion: (1) availability of an adequate population of osteogenic cells, (2) the presence of an osteoconductive matrix within the region where new bone tissue is desired, (3) osteoinductive signals within the graft site, (4) adequate local blood supply to support a bone healing response, and (5) local mechanical environment suitable for bone formation Fusion procedures are usually augmented with instrumentation to minimize the motion across an interbody bone graft, thus hopefully resulting in an increased fusion rate Heggeness tested the translaminar screw fixation Biomechanically in his study, using seven fresh cadaver spines with static eccentric loading and cyclic loading using servohydraulic material testing machine. The static eccentric loading test reveals that after instrumentation with translaminar screw fixation, the stiffness increased 2.4 times compared to the uninstrumented spines. Cyclic loading reveals that after 5000 cycles, the stiffness did not decrease significantly, concluding that the translaminar screw fixation can maintain the increased stiffness, thus improving the fusion rate In this case series, solid bony fusion and no apparent motion on the flexion-extension x-rays in the fused segment were documented in all patients. However, loosening of the screw was noted in one patient. However, no segmental motion could be detected on the flexion-extension radiographs. Heggeness in his study also reported that one case of loosening of the screw resulted in pseudoarthrosis, but the back pain in that case resolved in one year Implant failure was confirmed in one patient, which had three segments fixated despite there was no history of trauma after surgery. However, we found no neurologic deficit. Implant failure was also reported in Humke's study; He reported two from 173 cases who have implant failure on monosegmental fusion. Both patients did not show segmental motion on the flexion-extension radiographs, the screw fragments did not cause any symptoms, and no migration was reported In this study, there was no evidence of neurologic injury. According to other researchers who have described the same procedures, severe neurologic complications also not documented in conjunction with translaminar screws of the lumbar spine The minimal volume of screws may lower the possible danger of infection compared to transpedicular devices. This also leaves plenty of room for graft material to be placed, promoting firm bone fusion. Translaminar screws also use lower-cost implants compared to transpedicular screws The use of postoperative external immobilization is important to provide fusion with the correct position and minimalized the risk of implant failure and nonunion due to excessive loads on the implants, primarily in translaminar screw fixation Some experts believe that the risk of applying pedicle screw systems for degenerative lumbar and lumbosacral spine fusion is insufficient because of major anterior deficiency of the spine. The translaminar screw technique relies on an anatomically intact anterior column that can resist compression force. This is the reason why such a technique is ideal for degenerative cases presented in this study This study adds information that translaminar screw fixation can be an alternative to posterior stabilization, especially in degenerative lumbar disease. The limitation of this study is a single-center retrospective study and did not prospectively compare with other approaches, the size of the patient included in this study is small. Therefore, we came with case series, and the relevant data is sufficient to describe the functional outcome and fusion rates of translaminar screw fixation. For future studies, we recommend using a larger size sample or patients with more evaluation scoring and biomechanical test, if possible, compared to transpedicular screw fixation.4The enhancement of lumbar and lumbosacral fusion with the translaminar screw technique offers an easy way to improve quality of life, fusion rates and simplify postoperative management. This technique can be an alternative, useful, inexpensive and safe method for short segment fusions in patients with non-traumatic lumbar and lumbosacral spine problems.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not commissioned, externally peer-reviewed.This study has been reviewed and approved by the authors' Institutional Review Board.This Case report is not “First in Man” Study.Andhika Yudistira.Andhika Yudistira: conceptualization, writing original draft preparation, supervision, project administrator, validation.Maulana Hasymi Hutabarat: data collecting, data interpretation, writing original draft preparation, writing the paper and editing, validation.Lasa Dhakka Siahaan: data collecting, data interpretation, writing original draft preparation, writing the paper and editing, validation.Muhammad Alwy Sugiarto: data collecting, data interpretation, writing original draft preparation, writing the paper and editing, validation.We declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Histone acetylationis an important, reversible post-translationalprotein modification and a hallmark of epigenetic regulation. However,little is known about the dynamics of this process, due to the lackof analytical methods that can capture site-specific acetylation anddeacetylation reactions. We present a new approach that combines metabolicand chemical labeling (CoMetChem) using uniformly 13C-labeled glucoseand stable isotope-labeled acetic anhydride. Thereby, chemically equivalent,fully acetylated histone species are generated, enabling accuraterelative quantification of site-specific lysine acetylation dynamicsin tryptic peptides using high-resolution mass spectrometry. We showthat CoMetChem enables site-specific quantification of the incorporationor loss of lysine acetylation over time, allowing the determinationof reaction rates for acetylation and deacetylation. Thus, the CoMetChemmethodology provides a comprehensive description of site-specificacetylation dynamics. Histoneacetylations are hallmarks of epigenetic regulation and are closelylinked to the regulation of key cellular processes such as chromatinremodeling, transcriptional regulation, gene silencing, cell cycleprogression, apoptosis, differentiation, DNA replication, DNA repair,and nuclear import.5 An imbalance in histone acetylation has been associated with a varietyof human diseases including cancer,6 chronicinflammation,7 and neurological8 and metabolic disorders.9Living organisms arehighly dynamic systems and constantly exposedto external changes and strive to maintain homeostasis. Post-translationalmodifications (PTMs) play a crucial role in this process, as theyallow them to adapt to the protein function and activity in a rapidand highly dynamic manner. Histone lysine acetylation is a dynamicand reversible PTM regulated by histone acetyltransferases  and histone deacetylases.10 but the mechanisms and dynamicsby which they control each other’s stability and turnover arelargely unknown. An important reason for this gap in our knowledgeis the lack of adequate methods for the site-specific analysis ofacetylation dynamics and their interactions.Despite their central biological and medical importance, thedynamicsand site specificity of acetylation events at histones are still poorlyunderstood. Adjacent PTMs are generally accepted to influence oneanother,12 Moreover, while they allow toestimate the relative extent of a PTM under different conditions,immunochemical methods cannot be used to trace the turnover of PTMs.Mass spectrometry (MS) coupled to liquid chromatography (LC–MS)has emerged as a powerful method to investigate site-specific histoneacetylation,13 providing reliable, site-specificidentification and quantification of histone acetyllysines. MS-basedmethods also allow us to detect and quantify simultaneously occurringmodifications within the same protein sequence in a single experiment.Nevertheless, quantitative analysis of histone acetylation is particularlychallenging since the N-terminal histone tails are highly enrichedin lysine and arginine residues in a short stretch of amino acids.Trypsin, the protease of choice for histone analysis by LC–MSin a bottom-up approach, does not cleave at the C-terminal site ofacetylated lysines due to charge neutralization. Furthermore, theoccurrence of adjacent lysines and arginines may lead to random missedcleavage events, resulting in a variety of short hydrophilic peptides,which are difficult to retain on reversed-phase (RP) columns. Thereby,a nonhomogeneous pool of peptide species is formed, in which the sameacetylated residue can be present in different peptides. These chemicallynonequivalent species complicate the accurate quantification of thedifferent acetylated histone species due to their different retentiontimes and ionization efficiencies.15 To generatea more homogenous pool of acetylated peptides and to increase theretention of hydrophilic peptides during RP chromatography, primaryamino groups of extracted histones can be chemically propionylatedor acetylated at the protein level prior to tryptic digestion.18 Smith et al. introduced chemical acetylation of unmodified lysineresidues with deuterated acetic anhydride to quantify endogenous acetylationlevels of the histone H4 protein.16 Byderivatization with stable isotope-labeled acetic anhydride, the chemicallyacetylated lysines possessed the same ionization properties as theendogenously acetylated lysines. In another study, Feller et al. appliedstable isotope-labeled acetic anhydride to investigate the effectof HATs and HDACs on combinatorial histone acetylation profiles.19 The chemical acetylation of unmodified lysines,using stable isotope-labeled acetylating reagents, allows us to quantifyabundance levels of acetylated and nonacetylated histone species20 while retaining site-specific information.21Histone modificationsare still frequently detected using immunochemicaltechniques. However, site-specific antibodies against acetyllysinesoften lack specificity and cross-react with adjacent PTMs in the N-terminalhistone tails.22 To study histone acetylationdynamics, Evertts et al.23 and Zheng etal.24 introduced metabolic labeling usinguniformly 13C-labeled glucose ([U-13C]-Glc) as a metabolic precursor.[U-13C]-Glc is converted into 13C-labeled acetyl-CoA  at the protein levelprior to LC–MS analysis. Thereby, all lysine residues of thehistones are fully acetylated. Consequently, all peptide species generatedduring proteolytic digestion with identical amino acid sequences exhibitthe same chemical properties, permitting accurate relative quantificationby MS. Based on the mass shifts introduced by metabolic versus chemicallabeling, CoMetChem allows us to quantify site-specific reaction ratesof acetylation and deacetylation, a critical parameter to monitorthe dynamics of PTMs and the effects of pharmacological modulatorssuch as HDAC inhibitors.To tackle this issue, we developed a combined metabolic and chemicallabeling (CoMetChem) approach. CoMetChem combines [U-13C]-Glc-basedmetabolic labeling and chemical acetylation of nonacetylated lysineresidues with Supporting Information.Thechemicals used in this study are listedin the Supplemental Experimental Section.Experimentaldetails of the cell culture conditions can be foundin the 2, 1 mM CaCl2, 250 mM sucrose, 1 mM dithiothreitol(DTT), 1 mM phenylmethylsulfonyl fluoride, 10 mM nicotinamide, 10mM sodium butyrate, and 0.01% sodium deoxycholate (SDC)] was used.The homogenized cells were incubated for 10 min on ice and washedthree times with the nuclear isolation buffer without SDC. Histoneswere extracted from the nuclei using 0.2 M HCl. For more details,see the Supporting Information.For histone extraction, the cellnuclei were isolated first. The cells were collected and washed threetimes with PBS. For lysis, 500 μL of ice-cold nuclear isolationbuffer -Glc (13C2H3), respectively. The mass increments canbe resolved in state-of-the art TOF and orbitrap mass analyzers, andthe different isotopologues have the same ESI efficiencies. Completechemical acetylation of the nonacetylated lysine residues at the proteinlevel is essential for the CoMetChem strategy to ensure that onlychemically equivalent species arise from the tryptic digestion ofa given protein. We obtained an almost complete derivatization withboth TEAB and SB  asreaction buffers . The derivatizationefficiency was evaluated by LC–MS analysis of 14 histone lysineresidues including the H3(18-26) and H4(4-17) peptides harboring twoand four lysine residues, respectively. Acetylating lysine residuesthat are in close proximity to each other was particularly challenging.While both TEAB (99.9 ± 0.2%) and SB (99.8 ± 0.2%) resultedin a near-complete acetylation for the H3(18-26) peptide, which containstwo lysine residues , SB (97.9± 0.3%) resulted in a significantly higher acetylation efficiencyfor the four-lysine-containing H4(4-17) peptide compared to TEAB (96± 0.5%) . Thus, we continuedto use SB as a reaction buffer for chemical acetylation with 13C4,D6-AA.Unmodified lysine residues arechemically acetylated using stable isotope-labeled llysines 1A. These13C4,D6-AA, respectively. The macrophage cell line RAW 264.7was first cultured in [U-12C]-Glc-containing medium and then switchedto [U-13C]-Glc-containing medium (t = 0)  peptide species, generated by tryptic digestion from H3protein and covering a region with two adjacent acetylation sites(K18 and K23), were chosen as an example. Combined metabolic and chemicallabeling by CoMetChem results in nine different H3(18-26) peptideisotopologues . At t = 0, before the addition of [U-13C]-Glc,the following isotopologues can be detected  1B. Histopologues 1C. At thpologues 1D, as thpologues S2. At t 13C4,D6-AA-derived acetyl groups (m/z 540.8479).I: The nonacetylated H3(18-26)peptide which contains two 13C4,D6-AA-derived acetylgroup at K23 (II)and K18 (III), respectively. Both species are isobaric(m/z of 538.3353) and cannot bedistinguished at MS1.II, III: The endogenously single-acetylated H3(18-26) species,which contain a [U-12C]-Glc-derived acetyl group at K18 (II) or K23(III) and a m/z 535.8230).IV: The H3(18-26) peptide with two endogenous[U-12C]-Glc-derivedacetyl groups  isotopologues and K18 (VI), respectively. Both species are isobaric (m/z 539.3382) and cannot be distinguishedat MS1.V, VI: The endogenously single-acetylated H3(18-26) species whichcontain a [U-13C]-Glc-derived acetyl group at K18 (V) or K23 (VI)and a m/z 536.8262).VII, VIII: The endogenously double-acetylated H3(18-26)specieswhich contain a [U-13C]-Glc-derived acetyl group at K18 (VII) or K23(VIII) and a [U-12C]-Glc-derived acetyl group at K23 (VII) and K18(VIII), respectively, which are isobaric at MS1 (m/z 537.8306).IX: The endogenously double-acetylatedH3(18-26) species whichcontains two [U-13]-Glc-derived acetyl groups . In the case ofthe K18ac and K23ac positional isomers derived from [U-13C]-Glc , the b3 fragment ions contained either an acetyllysine derivedfrom [U-13C]-Glc for K18 (m/z 414.2593)or a chemically acetylated lysine for K23 (m/z 417.2828) . They7 fragment ions contained either a chemically acetylated lysine forK18 (m/z 777.4940) or an acetyllysinederived from [U-13C]-Glc for K23 .The single-acetylated species I and IIalong with V and VI, andthe metabolically double-acetylated species VII and VIII are positionalisomers and isobaric at MS1 1D but arm/z values, the stoichiometryof the K18ac and K23ac species can be determined based on the relativeabundance of the respective fragment ions.As the fragmentions differ in their stable isotopic compositionand their 25 Adetailed tutorial on bioinformatics workflow and data processing canbe found in the Supporting Information.Since chemical derivatizationleads to chemically equivalent isotopologues,it enables quantification of the relative abundance of the differentacetylated H3(18-26) peptides. However, another factor contributesto the complexity of the isotopologue mixture, namely, the naturallyoccurring 13C in the peptide backbone resulting in additional overlappingisotopologues at the MS1 level 1D. We thFigure S3), highlighting the tight control of the acetylationlevels at these residues.Comparison of the relative abundance levels of the different acetylatedH3(18-26) species revealed that the endogenously nonacetylated specieswas the most abundant (52.4%), followed by the single-acetylated K23acspecies (28.4%), the double-acetylated K18acK23ac species (14.2%),and the single-acetylated K18ac species (5.0%) 2A. The l24 where [U-13C]-Glc was used for metabolic labelingwithout additional chemical acetylation. Individual turnover rates(k) were calculated by fitting experimental valuesto exponential growth or decay functions to calculate the site-specifichalf-lives (t1/2 = ln 2/k)  2E. Thes ln 2/k) 1C, IV cak) was multiplied with the abundance (A) of the respective peptide species. The single-acetylated K23acand K18ac species showed a turnover of 3.07%/h and 0.38%/h  species. For this, the turnover rate  peptide are interconverted by at least 12 different reactions3.28 for histone methylation analysis. The strategy is basedon linear fitting of the initial interconversion fluxes, where thefluxes are the slopes of the time courses and thus represent the increaseor decrease in the different acetylated peptide species over time. This approach isbased on the simplified assumption that the substrate concentrationis initially relatively constant, and therefore, the correspondingproducts initially increase linearly with time. To estimate the acetylationand deacetylation rates (υ), the measured fluxes need to bedivided by the relative abundance of the respective substrate at t = 0. We determined site-specific reaction rates of acetylation and deacetylation  for the single-acetylated peptide.The acetylation rate at K23  was determined to be 0.019 ± 0.002/h, whereas the deacetylationrate at the same site was 0.059 ± 0.006/h. For K18, we obtained an acetylation rate of 0.0036 ± 0.0002/hand a deacetylation rate  of 0.032 ± 0.008/h. The comparison of the single-acetylatedH3(18-26) species revealed that the acetylation rate for K23 (a2)is approximately five times higher than the acetylation rate at K18(a1). The difference in site-specific deacetylation rates at K18 (d1)and K23 (d2) is not as pronounced as in the acetylation rates, butK23ac still showed a two times faster deacetylation rate comparedto K18ac. Thus, acetylation and deacetylation rates can substantiallydiffer between lysines within the same peptide.Since CoMetChem enables the direct comparison of the abundanceof all peptide species, we were able to determine individual reactionrates of acetylation and deacetylation for all H3(18-26) species.However, it is not possible to calculate rates for species that arenot present before label addition , it was approximately equal to the sum of the rates (d1and d2) determined for the site-specific deacetylation rates of thesingle-acetylated species . This indicates that in the H3(18-23) peptide, the site-specificdeacetylation rates were not affected by the acetylation state ofthe nearby lysine residue.As we cannotdetermine which of the [U-12C]-Glc-derived single-acetylatedspecies . Theyare thus two–three times faster than for the single-acetylatedpeptides. This indicates that an already existing acetylation promotesthe acetylation of the nearby site for the H3(18-23) species.In contrast to deacetylation rates,the CoMetChem approach allowsus to determine site-specific acetylation rates for the formationof the double-acetylated peptide. This is based on the conversionof the single-acetylated H3(18-26) species II and III into the double-acetylatedspecies VIII and VII by reactions a3 and a4, respectively 3. The siFigure S5A). We then quantified site-specific abundance levels forthe single-acetylated H4(4-17) species K5ac, K8ac, K12ac, and K16acto determine site-specific reaction rates for deacetylation and acetylation.The K16ac species showed, with 22.9%, the highest abundance of thesingle-acetylated H4(4-17) species . In contrast, very low abundances were observed for K5ac (1%),K8ac (1.5%), and K12ac (2.4%). Thus, site-specific reaction rateswere considered for the single-acetylated K16ac species only. Thesingle-acetylated H4(4-17)-K16 species showed a deacetylation rate(d4) of 0.048 ± 0.005/h at K16 and an acetylation rate (a4) of0.015 ± 0.001/h . Theseresults demonstrate that CoMetChem is applicable to determine thereaction rates of acetylation and deacetylation of different acetylatedhistone species. In this study, site-specific reaction rates of acetylationand deacetylation were determined for single-acetylated H4(4-17) species,as all isobaric isotopologue species could be distinguished at theMS2 level. To determine site-specific reaction rates for the double-acetylatedand three and four times acetylated H4(4-17) species, MSn experimentsare required to distinguish isobaric species.In addition, we determined site-specific reaction rates for thesingle-acetylated H4(4-17) species using CoMetChem. For this, we firstquantified the overall abundance level of the nonacetylated (43.5%),single- (27.8%), double-acetylated (16.1%), and three (6.2%) and fourtimes (6.4%) acetylated H4(4-17) species , which inhibits class I, II, andIV HDACs, and the benzamide derivative entinostat (MS-275)30 , whichinhibits the class I HDACs HDAC1 and HDAC3. RAW264.7 cells were firstcultured in [U-12C]-Glc-containing medium for 22 h, followed by apre-incubation of 16 h with either SAHA or MS-275 . Afterward, the culture medium was replaced with[U-13C]-Glc-containing medium including the corresponding HDAC inhibitor.We further exploredthe CoMetChem approach to investigate site-specific acetylation dynamicsupon pharmacological intervention with two HDAC inhibitors. We usedsuberanilohydroxamic acid (SAHA)Figure S5A). For the single-acetylated K16ac H4(4-17) species, treatmentwith MS-275 (20.1%) and SAHA (18.4%) resulted in significantly loweracetylation levels compared to the control (22.9%) . Although HDACi treatment did not result in higheracetylation levels for the single-acetylated H4(4-17) species, analysisof site-specific reaction rates revealed that treatment with MS-275and SAHA resulted in complete inhibition of deacetylation reactionsat K16 (d4) compared to the control  and to decreased acetylation rates at the same site .As expected, the HDAC inhibitors led to decreased deacetylation.For H4(4-17), this is reflected in the significant decrease in nonacetylatedspecies from 43.5%  to 18.0%  and 12.8%  and a significant increase in the two ,three , and four  times acetylated H4(4-17) peptide species. As expected, HDAC inhibitors also resulted in decreased deacetylation.This is reflected in the significant increase in the double-acetylatedH3(18-26) from 14.2 to 21.1% with MS-275 and to 28.6% with SAHA 4A. In adS8). The deacetylation ratefor the double-acetylated H3(18-26) species (d3) was 0.066 ±0.026/h with MS-275 and thus similar to the control (S9). In contrast, SAHA decreased the deacetylation rate from0.084 ± 0.027/h to 0.04 ± 0.02/h (S10). Acomparison of the site-specific deacetylation rates for the single-acetylatedH3(18-26) species revealed that MS-275 reduced the deacetylation ratesat K23(d2) from 0.059 ± 0.006/h to 0.020 ± 0.007/h and from0.032 ± 0.008/h to 0.012 ± 0.007/h at K18(d1). With SAHA,the acetylation at K18 appeared to be relatively constant, indicatinga near-complete inhibition of the deacetylation at this site. We note,however, that this requires further investigation, as the measuredvalues were at the detection limits of the method. The deacetylationrate at K23 was reduced from 0.059 ± 0.006/h to 0.020 ±0.005/h upon exposure to SAHA  species 4 and S8. control 4B and S9 to SAHA 4B. Treat to SAHA 4C.WhileTaken together, these results demonstrate that our CoMetChem approachprovides a comprehensive description of the dynamics of reversiblelysine acetylation by determining the site-specific reaction ratesof deacetylation and acetylation. Using CoMetChem, we were able todescribe the dynamic effects of HDACs in a site-specific manner usingHDAC inhibitors.13C4,D6-AA results in fully acetylated tryptic peptide isotopologues, whichare chemically equivalent. Thus, the isotopologues only differ intheir mass due to their isotopic composition while having the sameionization and ion transmission efficiencies during MS analysis. Dueto the chemical equivalence of the peptide species, site-specificabundance levels can be determined with high precision, and site-specificreaction rates of acetylation and deacetylation can be quantified.These reaction rates describe the acetylation dynamics much more accuratelythan half-lives and allow the kinetics of individual acetylation sitesto be directly compared. Thereby, CoMetChem goes beyond half-livesand turnovers, as site-specific reaction rates of acetylation anddeacetylation can be determined, allowing a comprehensive assessmentof the dynamics of this reversible modification. CoMetChem expandsthe repertoire of dynamic proteomics methods and represents a particularlyvaluable approach for deciphering the site selectivity of HDACs andprotein acetyl transferases, facilitating the development of HDACinhibitors with well-characterized substrate profiles.With the CoMetChem methodology,we present a novel approach forthe analysis of site-specific histone acetylation dynamics and reactionrates. Compared to the combinatorial use of metabolic [U-13C]-Glclabeling and chemical acetylation with propionic anhydride, the combinatorialuse of stable isotope-labeled metabolic precursors ([U-13C]-Glc) andchemical acetylation using


The Selfish Gene, Richard Dawkins (In his book  Dawkins  suggests Dawkins  into popEmmer wheat (Triticum turgidum), an allopolyploid with duplicated genomes from two progenitor species . Their focus was on the behaviour of the nucleoli that are responsible for rRNA transcription and, thus, can serve as a readout for metabolic activity  can be released when parts of the genome are absent. The most likely scenario would be an inhibition of the Einkorn NORs by factors located on the genome deriving from the wild ancestor of Emmer. Second, all the factors needed for the activation of a NOR are probably present in the immediate neighbourhood. Thus, the inhibition of nucleolar activity is acting over distance, while the activation is local.The first contribution by von Well et al.  asks theAlstroemeria), a very diverse group of lilies that occur in South America and Australia and are also of commercial interest due to their attractive and variable flowers. Two clades of this genus differ strongly with respect to heterochromatin content, indicating that there is active regulation of transcription activity between different chromosomes that are very asymmetric in this genus, making them especially suitable for cytogenetical approaches. The focus of the study was on the Brazilian species Alstroemeria longistaminea, which, in addition to its 2 n = 16 chromosomes, harbours an additional B chromosome. This type of chromosomes is thought to derive from hybridisation events and is found in many species. B-chromosomes behave as “genetic parasites”  object. If the factors required for silencing (in case of the Einkorn genome or the B-chromosome) or the factors required for autophagy  are limited in abundance, recruitment to a particular site would by itself result in the depletion from other sites. Mutual competition for a limited resource would then lead to patterning across the genome without the need of a superimposed “genetic identity”. Are these competitive interactions between different genetic loci the cellular correlate of the “invisible hand” that according to Adam Smith (1773) regulates the commonwealth and emerges from the economic competition between otherwise unrestrained individuals?What do the three contributions tell about genomic integrity? The wild B-genome in


Background: Despite the exhaustive body of literature on the demands of ice hockey, less is known about the relationships between functional performance testing protocols (on ice and off ice) and performance in a game situation. The objective of this review is to provide an overview of these associations. Methods: This review aims to identify on- and off-ice testing currently used in the scientific literature and their possible transfer to game performance as well as identifying research gaps in this field. Results: The 17 selected studies showed that off-ice and on-ice fitness test results can be modestly transferred to the player’s selection as well as global and advanced performance indicators. Conclusion: This review of the literature reinforces the importance of strength and conditioning coaches administering previously validated fitness tests. Regarding the academic research, it is also proposed to use performance markers that are directly related to the players’ on-ice performance to represent more accurately the relationship between the players’ fitness level and their work output. Three research gaps were also identified in relation to targeted populations, choice of performance markers and data measurement methods. Subsequent to the legendary Summit Series in 1972, ice hockey was recognized as an international sport, which resulted in an invitation to professional players to compete at the Nagano Olympics in 1998. In 2020, over 1.6 million players took to the ice in more than 70 countries 3], and BTechnical components refer to specific ice hockey skills. Skating speed  and skatGenerally speaking, hockey organizations  aim to identify players with the greatest potential and develop them to meet the highest possible standards. Talent identification is a challenging process that includes several approaches. At the professional level, scouts play a major role in evaluating and ranking players in order to select the best prospects. In scouting, most evaluations derive from the scouts’ subjective judgment and observation . While tA second approach to assessing players’ talent or potential and development is based on objective approaches such as testing. Here, players must perform different tests aimed at evaluating their level of ability regarding specific components of performance. Testing or measurements can be conducted in three specific contexts: off ice, on ice, and game settings. Despite this objective method of measuring a player’s level of performance, these approaches have their limitations. Over the years, fitness assessment in ice hockey has been used often and for multiple purposes. In this regard, Quinney et al.  demonstrDespite the usefulness of functional performance tests, their validity for predicting on-ice performance has not yet been established. The relationship between fitness testing and actual in-game performance has been studied in sports such as rugby  and soccThanks to over fifty years of research, we have a good understanding of the physical demands of hockey and the ways to effectively assess players’ physical attributes. Nevertheless, the question of whether or not these assessments systematically translate to or predict game performance remains unclear. In this regard, we observe a knowledge gap with regard to establishing associations between fitness testing and performance outcomes.This scoping review has two objectives. First, it aims to identify the studies investigating associations between ice hockey players’ fitness level and player selection , global performance indicators  and advanced indicators . The second objective of this scoping review is to identify research gaps in the literature with regard to fitness indicators and on-ice performances in organized hockey. The goal is to summarize our findings and share them with practitioners and scholars interested in learning more about the associations between fitness testing and game performance.In view of our objectives and the absence of a systematic review of the subject, we considered the scoping review the methodology best suited for the study . The revNext, and with the assistance of a university librarian, we searched four different databases  for relevant studies published between 1980 and 2021 using the keywords: “Ice hockey” WITH “predictor-determinant” OR “power” OR “Strength” OR “Endurance” OR “Speed” OR “ Aerobic” OR “Anaerobic” OR “Agility” OR “Fitness” OR “Performance.” We also did a manual search of the grey literature using similar keywords and, finally, another manual research of the lists of references in each identified study.We extracted references and exported them to Endnote 8 software. To define the inclusion criteria for the articles in our scoping review, the PICO framework was used . ArticleAfter all relevant studies had been chosen, they were screened by at least two authors to ensure they aligned with the research question. Whenever reviewers disagreed about an article’s relevance, a third reviewer was called on to settle the argument. The data from all studies included were then charted by three authors. V.H.P. extracted data from 90% of the studies and A.P. subsequently extracted data from the remaining 10%. A.P. and J.G.R. checked 20% of V.H.P.’s data extractions for accuracy and then screened the studies using the same process. Any discrepancies were discussed by the authors during their weekly group meeting. Agreement was 90% regarding the studies’ inclusion/exclusion. The three authors  discussed the content of the studies on the Excel form to highlight relevant relationships between ice hockey testing and performances.We analyzed the strength of associations between fitness testing protocols and game performance by taking a deeper look at the statistical outcomes of each study. Because of the different statistical procedures  used in each study, findings were interpreted in different ways. When possible, we interpreted the magnitude of associations by analyzing the effect sizes from each analysis. For these interpretations, we used Cohen’s  guidelinOf the 2364 papers gathered from the search strategy, 259 abstracts and titles (11%) met our inclusion criteria see . After tSeven studies 41%) combined both on-ice and off-ice tests in their designs ,36,37,38% combineFor on-ice testing , all stuThe three main categories of performance markers used in the selected studies refer to players’ global performance, the metrics associated with specific performance, and team selection, talent identification and/or draft status. For global performance, statistics regarding the number of games played, ice time (playing), and total points  were those reported in three studies (18%) ,39,42. ADespite the abundant scientific literature in the ice hockey domain, we have to recognize that most studies aimed at establishing physiological demands of ice hockey ,3,4,5 anThe selected papers showed that the physical attributes that are the more frequently used for off-ice testing are muscular strength, lower limbs’ power and anaerobic capacity. Such attributes are congruent with ice hockey’s physiological demands and refers to key fitness components that might be linked with excellence in ice hockey ,5. NeverFor on-ice measurements, we observed less variability in the choice of testing protocols. Interestingly, skating speed ,48, agilThe second objective of this scoping review was to identify key research gaps in the area of functional performance testing in ice hockey. The first research gap we have identified is in regard to the selected populations. Most of the selected studies refer to junior, college-varsity and national team players, and very few studies examined female and youth (U18 and younger) hockey. Even if some recent publications focus on female hockey ,52,53, tA second research gap in the domain of performance assessment in ice hockey is the choice of performance markers, and the methods used to collect such indicators. The recent emergence of hockey advanced analytics and the different new categories of performance indicators is a promising area of development because they allow researchers to test multiple hypotheses in relation to gameplay performance and see how physical, technical, and psychological attributes can determine how players perform in specific aspects of the game. From this perspective, we think that quantitative game context indicators that can catch specific and measurable attributes would be helpful for authors who aim to explain ice hockey performance. As a third research gap, we think that the methods of data collection need to go further by taking account of more objective indicators instead of considering indicators that are simpler (like draft rankings), which might not be related to the players’ attributes. In this regard, some validation  studies are needed to make sure that the objective, advanced indicators are congruent with the attributes under study.A final research gap concerns data collection methods. Many of the reported papers used systematic observation, box scores analyses, and data collected from video observations. Even if most of these methods are valid, they have some limitations regarding the quantity of information that they provide for researchers. Therefore, the recent emergence of artificial intelligence-based systems or advanced analytics platforms  could be a solution for such methodological shortcomings by offering the opportunity to create and analyze large and deep datasets. In addition, we think that using these technologies involving global and local positioning systems (GPS-LPS) that were recently used in the exercise physiology domain ,56,57 woThis scoping review provided an overview of the current state of research regarding ice hockey performance assessment. The main contribution of this study was to observe the relevance of multiple testing approaches when it comes to the athlete’s follow-up, team selection, talent evaluation, and game performance analyses. In this regard, the present review provides some justification for the usefulness of a different testing approach to establish standards and norms in different playing levels. In a practical way, this review provides interesting insights for coaching staffs, strength conditioning coaches, and professionals involved in talent identification . For coaching staff and scouts, having a better understanding of the association between their players’ physical attributes profile and the potential outcome during competition might guide them to adjust their rosters for specific events. For strength and conditioning coaches, knowing specifically which functional performance indicators to develop in relation to specific game situations is helpful in designing efficient training programs at different periods of the year.Despite its contribution, this review also has its limitations. The selected papers came from scholarly journals (written in English language), and some works from the academic domain (like thesis) might not have been selected for this review. In this regard, it is plausible to think that some “unpublished” protocols exist, which might have led to additional observations in this review. We think that during the next decade, new technologies might appear and help assess players physical fitness and on-ice performances. Combining such instrumentation will lead to stronger and more conclusive evidence between functional performance and their outcomes during competition. Another limitation of this paper is that it focuses only on one aspect that can contribute to players’ success on the ice, which is physical fitness. Other technical , tactica