N,N′-methylenebisacrylamide with a purposely designed bis-sec-amine monomer, namely, tetraethyl)bis(azanediyl))bis)bis(phosphonate) (PCASS). The aim was to ascertain whether the introduction of phosphonate groups, well-known for inducing cotton charring in the repeat unit of a disulfide-containing PAA, increased its already remarkable flame retardant efficacy for cotton. The performance of M-PCASS was evaluated by different combustion tests, choosing M-CYSS, a polyamidoamine containing a disulfide group but no phosphonate groups, as a benchmark. In horizontal flame spread tests (HFSTs), M-PCASS was a more effective flame retardant than M-CYSS at lower add-ons with no afterglow. In vertical flame spread tests, the only effect was afterglow suppression with no self-extinguishment even at add-ons higher than in HFSTs. In oxygen-consumption cone calorimetry tests, M-PCASS decreased the heat release rate peak of cotton by 16%, the CO2 emission by 50%, and the smoke release by 83%, leaving a 10% residue to be compared with a negligible residue for untreated cotton. Overall, the set of results obtained envisage that the newly synthesized phosphonate-containing PAA M-PCASS may be suitable for specific applications as flame retardant, where smoke suppression or reduction of total gas released is a key requirement.A novel polyamidoamine (M-PCASS) bearing a disulfide group and two phosphonate groups per repeat unit was obtained by reacting  Cellulosic materials are among the most flammable materials for domestic use ,2. Regar® by Solvay Group, Bruxelles, Belgium) and N-methyloldialkyl phosphonopropionamides  have been the main FRs for cotton used over the past 40 years [Impregnation with solutions of flame retardant (FR) substances is a common practice to overcome this drawback . Histori40 years ,13. Howe40 years , hence i40 years . It has 40 years , thanks 40 years .prim-monoamines, including naturally occurring α-amino acids or bis-sec-amines with bisacrylamides. PAAs are structurally highly versatile, can be engineered to be biodegradable and biocompatible, and, in the past, have mainly been investigated as potential biomedical polymers [N,N′-methylenebisacrylamide (M) with glycine (M-GLY) and most other natural α-amino acids were particularly effective in this regard [L-cystine with N,N′-methylenebisacrylamide (M-CYSS) effectively extinguished the flame in VFSTs as well [Polyamidoamines (PAAs) are a family of highly versatile multifunctional polymers obtained, in linear form, by the aza-Michael polyaddition of polymers ,17. In tpolymers ,19,20. Is regard . As a rus regard , but, in as well .Given these premises, in this work, it was thought to be worth studying whether, similarly to disulfide groups, the addition of phosphonate groups in PAA repeat units could increase their efficacy as FR for cotton by synergistically improving the intumescence of PAAs and favoring cotton charring.To this purpose, a novel PAA was prepared and tested, namely, the aza-Michael polyaddition products of M with tetraethyl)bis(azanediyl))bis)bis(phosphonate) (PCASS), henceforth referred to as M-PCASS, whose repeat unit contained four phosphonic ester groups and one disulfide group. In this work, the effectiveness of M-PCASS as FR for cotton was investigated by both HFSTs and VFSTs, as well as oxygen-consumption cone calorimetry tests. The aim of this paper is to report on the results obtained.N,N′-methylenebisacrylamide , L-cystine , lithium hydroxide monohydrate , 1 M hydrochloric acid (HCl), cystamine dihydrochloride , triethylamine , diethyl ether, dichloromethane, and anhydrous sodium sulfate (Na2SO4) were supplied by Sigma-Aldrich . Diethyl vinylphosphonate (97%) was supplied by TCI Chemicals (Israel) and used as received. Cotton (COT) with an area density of 240 g∙m−2 was purchased from Fratelli Ballesio S.r.l. .−1 ; samples (circa 5 mg) were placed in open alumina crucibles, in either inert or oxidative atmospheres, under 20 mL∙min−1 gas flow.Thermogravimetric analyses (TGA) of PAAs and PAA-treated cotton fabrics were performed in inert (nitrogen) and oxidative (air) atmosphere on a TAQ500 thermogravimetric balance from 50 to 800 °C at a heating rate of 10 °C∙minThe surface of untreated and PAA-treated cotton fabrics was analyzed by an EVO 15 equipped with a ULTIM MAX 40 probe scanning electron microscope (SEM)  operating at 8.5 mm working distance, under a 5 kV beam voltage, equipped with Energy-Dispersive X-ray Spectroscopy  to perform elemental analyses. A fabric piece (5 mm × 5 mm) was fixed to a sample holder and then gold metallized.2O (2 mL) and, then, keeping the solution in an ice bath, triethylamine  was slowly added. The mixture was warmed up to room temperature, stirred for 15 min, then a solution of diethyl vinyl phosphonate  in H2O (2 mL) was added. Once the addition was finished, the reaction mixture was left under stirring at room temperature for 48 h. The solution was treated with diethyl ether (5 mL), and the product was extracted from the aqueous one with dichloromethane (2 × 5 mL) after phase separation. The combined organic phases were dried over Na2SO4, filtered, and the solvent removed under vacuum to give a pale-yellow oil. Yield: 0.69 g, 73%. The reaction scheme is shown in 1H-and 31P-NMR )bis(azanediyl))bis)bis(phosphonate) (PCASS) was synthesized, optimizing the procedure previously reported . In brie 31P-NMR .Synthesis of M-PCASS. N,N′-methylenebisacrylamide, M,  and PCASS  were dissolved in water (3.00 mL) at 45 °C. After complete monomer dissolution, the reaction mixture was cooled to room temperature and gently magnetically stirred at room temperature for 8 days in the dark. Then, it was diluted with H2O (30 mL) and the pH adjusted to 4.0 with 1 M HCl. The product was finally retrieved by freeze-drying. The yield was nearly quantitative. The reaction scheme is shown in Synthesis of M-CYSS. M-CYSS was synthesized following the procedure already reported [N,N′-methylenebisacrylamide , L-cystine , and lithium hydroxide monohydrate  were dissolved in H2O (7 mL) and heated under stirring to 45 °C until the complete dissolution of the monomers occurred. The reaction mixture was cooled to 25 °C and left at this temperature for 3 days in the dark, then it was diluted to 70 mL with water, and the pH was adjusted to 9.2 with a 1 M HCl solution. The product was finally retrieved by freeze-drying. The yield was nearly quantitative. The reaction scheme is shown in reported . In brie1H-NMR and FT-IR/ATR spectroscopy  were determined by weighing each sample before (iW) and after drying following impregnation (fW). The add-ons were calculated according to Equation (1):Strips of cotton fabric of a 30 mm × 60 mm size were dried by heating at 100 °C for 2 min and then weighed. Subsequently, they were impregnated twice with aqueous solutions of PAAs of suitable concentration, drying for 3 min at 100 °C after each deposition. The total dry solid add-ons  and Vertical flame spread tests (VFSTs) were carried out by applying a 20 ± 5 mm long butane flame to the short side of 30 mm × 60 mm specimens according to the ISO 3795  and ISO −2 irradiative heat flux of square fabric samples (100 mm × 100 mm) was investigated using an oxygen-consuming cone calorimeter . Measurements were carried out in a horizontal configuration following a procedure previously reported [−2), total heat release , and residual mass fraction  were determined [2∙m−2), carbon monoxide (CO), and carbon dioxide (CO2) yields, both expressed in kg−1, were also determined. Prior to the combustion tests, all specimens were conditioned to constant weight at 23 ± 1 °C for 48 h at 50% relative humidity in a climatic chamber. Each experiment was performed in triplicate and the standard deviation calculated.The resistance to a 35 kW∙mreported , optimizreported . Paramettermined . Total SIn this work, the efficacy of the PAA coded M-PCASS a as a flN,N′-methylenebisacrylamide with L-cystine, as already reported )bis(azanediyl)) bis)bis(phosphonate) (PCASS) monomer .The TG thermograms of M-PCASS in nitrogen and in air in the 50–800 °C range were compared with those of M-CYSS under the same conditions a,b. The The higher thermal stability of M-PCASS and M-CYSS in air, with respect to that observed in nitrogen, was ascribed to their ability to form an intumescent and thermally stable char, as already observed for other PAAs ,19,20,282), 1375 (δ C–H), 1320 (δ O–H), and 1015 cm−1 (ν C–O) for cellulose; 1651 (ν C=O) and 1540 cm−1 (δ N–H) for PAA; and 1215 (ν P=O) and 790 cm−1 (ν P–C) for the phosphonate group.Untreated and PAA-treated cotton fabrics  were characterized by FT-IR/ATR spectroscopy . All speThe morphologies of the COT/M-PCASS samples were analyzed by SEM d–f and cThe EDX analysis of COT/M-PCASS  revealedonset10%, Tmax, and RMF values are reported in onset10% and Tmax values , Vertical Flame Spread Tests (VFSTs), and oxygen-consumption cone calorimetry tests. The results were compared not only with those of COT but also of COT/M-CYSS, whose performance in this respect had already been studied .COT and PAA-treated cotton fabrics were tested in a horizontal configuration, applying a butane flame for 3 s. COT/M-PCASS suppressed the flame at an 8% add-on, leaving an RMF of 81%, whereas COT/M-CYSS blocked the flame only at an add-on above 12%, leaving an RMF of 93%, ) and during the flaming phase (TSR(FP)), CO and CO2 emission yields have been collected and reported in (NFP) and TSR(FP) values  values  and the ) values c. Notice2 emission is much more significant .In previous work , it shouL-cystine with N,N′-methylenebisacrylamide, proved active as a flame retardant of cotton also in the severe vertical flame spread tests [Several polyamidoamines that have been shown to be able to protect cotton from combustion in horizontal flame spread tests by sensitizing cotton to thermal decomposition and promoting its intumescence in the temperature range of 350–450 °C. In addition, disulfide-containing polyamidoamines, including M-CYSS, obtained by the reaction of ad tests , possiblad tests , due to ad tests . Their mN,N′-methylenebisacrylamide was reacted with tetraethyl)bis(azanediyl))bis)bis(phosphonate), coded PCASS, to give a new polyamidoamine, coded M-PCASS, bearing two diethylphosphonate groups and a disulfide group per repeat unit. It should be noted that this is the first time ever that polyamidoamines-containing phosphonate groups have been investigated as flame retardants.The aim of this work was to ascertain whether the introduction of phosphonate groups in disulfide-containing PAAs could increase their already high efficacy as flame retardants for cotton. To this purpose, M-PCASS was applied as a coating on cotton fabrics and its flame-retardant efficacy, evaluated by several combustion tests, choosing the polyamidoamine coded M-CYSS, which contains disulfide groups but no phosphonate groups, as a benchmark. In horizontal flame spread tests, both M-PCASS and M-CYSS suppressed cotton combustion, but the former was more efficient than the latter. Specifically, the minimum M-PCASS add-on required for extinguishment was 8%, to be compared with a minimum M-CYSS add-on of 12%. Moreover, in contrast to what was observed in the M-CYSS-coated cotton, no afterglow was observed in the M-PCASS-coated cotton. On the other hand, in vertical flame spread tests, M-CYSS extinguished cotton combustion at a 16% add-on, whereas M-PCASS did not.Morphological analysis of the burnt areas of M-PCASS-treated cotton fabrics subjected to horizontal flame spread tests demonstrated that the polyamidoamine coating bonded the cotton fibers upon melting.2 emissions were halved.Oxygen-consumption cone calorimetry tests showed that M-PCASS decreased the HRR peak of cotton by 16%, in addition to generating an RMF of 10.0%, to be compared with the RMF < 1% of untreated cotton. Noticeably, the M-PCASS coating was able to decrease the smoke release by 83% and, in addition, COThe set of results collected in this work allows us to formulate initial interesting considerations regarding the benefits deriving from the introduction of phosphonate groups in the repeat units of disulfide-containing polyamidoamines. It may be observed that they gave rise, overall, to appreciable improvements in horizontal flame spread tests, although they could not suppress cotton combustion in the severe vertical flame spread tests. Additionally, they induced a dramatic reduction in gas and smoke release, which endowed them with a potential as flame retardant in specific fire scenarios where the fume and gas abatement was a stringent requirement. This points to the suitability of carrying out further studies on the flame-retardant properties of polyamidoamines containing phosphonate groups with different chemical structures and architectures.


ActivePresenter v9. Atomi Systems, Inc, Headquarters: No 281, Group 1, Phu Dien ward, North Tu Liem, Ha-noi, Vietnam; support@atomisystems.com; https://atomisystems.com/download/; $0 - $399 depending on license selected. Technical requirements: Operating Systems: Windows 8.1, Windows 10, Windows 11 (only 64-bit is supported), macOS 10.15, macOS 11, macOS 12; Hardware: 2 GHz or faster processor with SSE2; greater than 4 GB of RAM; 4 GB of available hard-disk space for installation; microphone. While there are a variety of free applications and software available for video recording and/or editing, ActivePresenter provides a single authoring tool for developing online learning objects presented in a familiar interface. For the purposes of this review, online learning objects are broadly defined as: “digital materials…with defined instructional value that can be used in a variety of ways to improve teaching and learning” . This isActivePresenter has three versions: Free ($0), Standard ($199), and Pro ($399). The free version can be downloaded to multiple devices and is intended for non-commercial use only. The Standard and Pro version licenses cover a single download for one device and includes perpetual access to the purchased version as well as any future software updates. Additionally, there is an educational discount program offering reduced individual Pro licenses ($199) and discounts for Pro educational licenses purchased in bulk.The free version of ActivePresenter is surprisingly robust for screen capture/video recording, editing, and video publishing options. The following sections will detail each of these three areas.Like other screen recording tools, ActivePresenter allows users to record the full screen or adjust the recording area to the desired size. The microphone can also be turned on or off providing the flexibility to either record audio live or add narrations later during the editing process. If a camera is available, it can be turned on/off to include a talking head with the screen capture or just to record the screen only. In addition, ActivePresenter imposes no limits on the number of videos that can be recorded. There are also no time limits on the length of the recording, compared to other free video recording tools, such as Loom or ScreenPal (formerly Screen-cast-O-Matic), that do impose time limits.The major advantage to using ActivePresenter is that it has a simple learning curve. ActivePresenter mimics the Microsoft Office layout so no matter the level of experience with video editing, the user can easily recognize the layout and find their way around the product. Once a video is recorded, it is opened in the ActivePresenter interface for editing. The figure below highlights the major areas of the ActivePresenter interface.The video and audio editing features included in the free version are: cutting/copying/deleting sections of recorded video/audio, splitting or combining video/audio files, importing other video/audio files, and inserting closed captioning. Closed captions have to be manually entered in the free version, so it is recommended that videos be uploaded to YouTube or other sharing platforms to utilize their automatic speech-to-text closed captioning.ActivePresenter can also be used to create professional online learning objects by combining screencast/video recordings and PowerPoint slides. The recording is treated as a single “slide” in ActivePresenter as if in the PowerPoint environment. Existing PowerPoint files can be imported into ActivePresenter or new slides can simply be added to accompany the video, such as an introduction and ending slide. In addition, other familiar PowerPoint features are included such as adding shapes , inserting animations and transitions between slides , changing slide layout and colors, and more (example video ). Just nFor video publishing, the free version of ActivePresenter has two options: 1) exporting to an image file, or 2) exporting to a video file . These two options do not include a watermark, however those may appear when attempting to export file types from the free version that are only available in the Standard or Pro versions (e.g. HTML 5 preview). The exported video file can then be uploaded to YouTube or another platform for publishing. For librarians that casually create video tutorials, the free version of ActivePresenter is likely sufficient for recording and editing needs. However, the Pro version should be considered for librarians who frequently develop learning objects and online tutorials, or record lectures. In particular, if the intent is to integrate content into learning management systems, the Pro version includes the ability to export learning objects to HTML 5 and to Sharable Content Object Reference Model (SCORM) 1.2, SCORM 2004, and xAPI. For a comparison of the features between the three versions, visit their website .Two comparable products to ActivePresenter that offer screen recording, video editing, and publishing in a single authoring tool regardless of operating system are ScreenPal (previously Screencast-O-Matic) and Camtasia , 5. As iActivePresenter is a recommended tool for librarians and information professionals who occasionally or regularly develop online learning objects including instructional tutorials, modules, and lectures, as well as embed learning objects into web pages or learning management systems. Depending on the extent of work in creating these materials, a paid version to ActivePresenter might be more appropriate to consider. Beyond libraries, it can also be recommended to faculty for use in online teaching, or students for class projects.


The tactical cooperation for the optimal interaction of team members is an essential performance-determining variable in sports games. The underlying cognitive memory structures of cooperative tactical actions have so far been little researched. Therefore, this study investigated the cognitive memory structure of tactical knowledge of handball actions in teams of different expertise and age groups. In the first experiment, tactical mental representation structures (TMRS) of 30 adult handball players of two different level of expertise were investigated. In the second experiment, TMRS of 57 youth handball players from three age levels were investigated. In both experiments the TMRS was measured with the structure dimensional analysis of mental representation (SDA-M) method. The SDA-M commences with a splitting procedure of a given set of concepts and reveals with a cluster analysis the relational structures of the concepts on an individual and a group level. Experiment one revealed that the TMRS differed significantly between skilled either/or less experienced handball players. Skilled handball players showed a hierarchical organized representation that shared more features with the basic tactical structure of the handball game than less experienced players. The second experiment revealed age-related differences of the TMRS between the age groups of the U15, U17 and U19 teams. Further data analysis revealed significant differences of the TMRS between experienced and less experienced handball players and between local and regional competition level players. We conclude that our current findings suggest that tactical expertise is mediated by elaborate cognitive tactical knowledge in memory. Furthermore, our results indicate that tactical knowledge plays a substantial role during tactical skill learning as it differs as function of age, experience, and competition level. From this point of view, team representations of game situations can be seen as a crucial factor for efficient and common interaction in fast-paced team sports. The interaction of team members represents a significant performance-determining factor in competitive team sports , 2. The However, when considering the expertise-dependent cognitive aspects of tactical behavior, we should first consider fundamental research of expertise in sport. A wide-ranging amount of research exists on perceptual-cognitive expertise in sport, comparing performance of experts, skilled athletes, and novices , 10. TheAlthough the research of tactical knowledge provides insights into details of knowledge representation, the applied methods do not deliver detailed structure of representations for tactical decisions. However, research on cognitive structures of tactical knowledge revealed expertise-dependent differences in representational structures in soccer  and futsIn line with these results, Nevett and French  and McPhIn both experiments, participants received no financial compensation for the participation and provided written consent prior to the experiment. Prior the experiment 2, informed consent of children’s parents was obtained. The study was approved by the Ethics Committee of Bielefeld University.In the first experiment, mental representation structures of adult handball players of two different level of expertise were investigated with the SDA-M. Based on previous reports , 22 moreth and 8th division. Prior both experiments, we determined the number of participants by a qualitative analyses of sample sizes that included invariance analysis results in the field of SDA-M studies.Thirty males participated in this study and were classified into two groups. The “skilled players”  were high-level handball players, playing in the third German Handball League. The skilled players mean age was 23.2 years (SD = 3.8), with a mean of 16.8 years of experience in handball and an average of 3.4 training hours per week (SD = 0.9). Three players played already in the first divisions of the German league and the SEHA-League, respectively. In addition, one player already played in the EHF-European-League. The “less skilled players” group  were experienced handball players with a mean age of 25.3 years (SD = 5.9), with an average of 1.8 training hours per week (SD = 1.1) and 17.1 years of handball experience from the 7Participants of each group sat in front of a presentation, five meters away from a screen (size 2.5 x 2 m). They confirmed verbally that they could see all details on the screen. All participants had a pencil and a board with sheet to judge the presented pairs of handball situations. The stimuli consist of twelve images that were always presented as a pair. One example pair is displayed in The stimuli entailed of twelve images that displayed indoor handball game situation. The design of the visual stimuli was inspired by the experiment of Lex and colleagues  and adapThe handball game is characterized by recurring game phases that occur in a different order depending on the course of the game . Within age = 41.2 years, SD = 8.4) with 17.4 years (SD = 9.3) coaching experience and trainer licenses c (n = 1), and b (n = 4) from the German Handball Association rated handball tactics (n = 28) regarding the correct and clear display of the team tactics . Fleiss’ Kappa [In our experiment, we applied twelve stimuli consisted of the four distinctive tactic situations that are aligned to four game phases in indoor handball. In an evaluation study prior the experiment, five handball coaches  on the right side. Every image was one time in the anchor position. Afterwards, the experimenter transferred the data into the SDA-M software QSplitV17.crit = 4.55, there was a significant difference between cluster solutions when λ < λcrit = 0.68 [The SDA-M method consists of four steps (detailed description in ). Initiat = 0.68 . Furthert = 0.68 , 47. TheIn the second experiment, TMRS of youth handball players from three age levels were investigated with the SDA-M. Based on previous reports  we expecage = 15.2 years, SD = 2.4) participated in this study. The players regularly competed in the youth league of their age group  on a local (n = 31) or regional level (n = 26). The mean age of the U15-group (n = 18) was 12.2 years (SD = 0.9), 15.6 years (SD = 0.5) for the U17-group (n = 22), and 17.8 years (SD = 0.7) for the U19-group (n = 17). The youth handball players had 1–5 years (n = 18), 6–9 years (n = 18), and ≥10 years (n = 16) handball experience and an average of 3.2 (SD = 1) training hours per week.Fifty-seven male youth handball players  and transition to attack (item 5). Thus, skilled and less skilled handball players had different tactical representations. Invariance analysis revealed that the representation structure of the two groups were significant different λ = 0.53 < λcrit = 0.68. Additionally, comparing the structures of both groups to the ideal structure, analysis indicated that skilled handball players structures shared more similar features with the ideal structure (ARI = 0.65), then less skilled player (ARI = 0.27). In sum, data of experiment one revealed that the TMRS of skilled handball players showed a hierarchical organized representation with clusters that represent team tactics in handball. In contrast, amateurs showed less organized TMRS.Representation structures for the skilled and less skilled handball players are displayed in crit = 4.55 for a given significant level of α = 0.01. In contrast, the cognitive structure of the U17 and U19 showed distinct clustering of team tactics. The dendrograms of the U17 showed three distinctive clusters. However, two items (4 and 11) can be considered as singles, as the links to the other items were above the critical value. The cognitive structure of the U19 showed three distinctive clusters and one single (item 5), two clusters were identical to the clusters of the ideal solution. The invariance analysis revealed significant differences between the cluster solutions of the three age groups (λ < λcrit = 0.68), U15—U17 λ = 0.42, U15—U19 λ = 0.43, U17—U19 λ = 0.54. Furthermore, the ARI revealed more similarity between the U19 dendrogram and the ideal structure (ARI = 0.64), then the U17 (ARI = 0.49), and the U15 (ARI = 0.06)  .crit = 0.68). No differences were found between the 6–9 and the ≥10 years’ experience group (λ = 0.71). In addition, the ARI revealed more similarity between both the 6–9 years’ (ARI = 0.67) and the ≥10 years’ experience group (ARI = 0.75) to the ideal structure, then the 1–5 (ARI = 0.61) years’ experience group  are displayed in ce group .crit = 4.55 for a given significant level of α = 0.01. In contrast, the cognitive structure of the regional group showed three distinct clusters and one single (item 4) of team tactics. The invariance analysis revealed significant differences between the cluster solutions (λ = 0.58 < λcrit = 0.68). Furthermore, the ARI revealed more similarity between the regional group dendrogram (ARI = 0.58) compared to the local group dendrogram (ARI = 0.39)  are displayed in  = 0.39) . SummariThe present study revealed in experiment 1 differences in the TMRS of skilled and less skilled adult handball players. In experiment 2, youth handball players showed age-, experience-, and expertise-depended differences in the TMRS. Research of TMRS in soccer revealed differences of skilled and less skilled adult players as well, but it was unclear whether expertise or experience made the differences. Thus, the results of this study are consistent with earlier tactical representation research in soccer . In compAs predicted, we found no functional cognitive structures of team tactics in young (U15) handball players. Consistent with predictions, youth U17 and U19 players’ representation structures were increasingly more similar to the ideal structure. Further, we found differences in the cognitive structure between handball beginners (1–5 years of experience) and intermediate experienced players (6–9 years), but no difference between the intermediate and the experience players (≥10 years). Moreover, youth handball players that played at a local level showed no meaningful clustering. As predicated, regional playing youth handball players showed a more functional TMRS that shared more features with the ideal structure, compared the local playing handball players. The current findings suggest that age, higher competition level and, especially in the beginner phase, more years of practice, play a crucial role in for sophisticated TMRS. However, the invariance of the cognitive structures from intermediate to high experience could be a hint that experience forms the cognitive representation in the beginning of tactical learning but should also be aligned with meaningful  experience , but ratFurthermore, the comparison between the cognitive structure of youth (Experiment 2) and adults (Experiment 1) revealed that youth handball players do not possess as elaborate TMRS as adult skilled, and less skilled players. This result is in line with findings from tennis  and baseAlthough more research is warranted, our findings indicate that expertise TMRS are well-integrated functional networks that reflect the functional structure of the team tactics. Especially, the team tactic transition to offense was not well integrated in less skilled and youth players. Thus, from an applied perspective, special training methods can be used to force elaborate TMRS. For instance, handball teams should not practice defense drills individually, instead the transition to offense should be integrated as often as possible. Thus, the goal of the defense actions would shift from stopping the opponent to intercepting the ball.One limitation of our study is that the current findings are reduced to the selected team tactics. Thus, to investigate sport overarching effects, future research could integrate more basic tactics that are applicable in various sports. First, this would provide evidence to advance theory about the cognitive structure of team tactics. Second, the influence of cognitive tactical structure on sport overarching transfer effects could be investigated . Thus, fIn conclusion, the present study gave insides in the cognitive representation of handball team tactics in both athletes and youth players. Results suggest that expertise is mediated by elaborate cognitive structures of handball tactics. Furthermore, current findings indicate that cognitive tactical structures differ as a function of age, experience and competition level. We assume that building individual and team representations of game situations are crucial factors for efficient and common interaction in fast-paced team sports. Therefore, identifying practices and methods that stimulate higher order processing in team cognitions could be a sophisticated approach to understand the nature of tactical representation and sport expertise.S1 File(XLSX)Click here for additional data file.S2 File(XML)Click here for additional data file.S3 File(XML)Click here for additional data file.


Videos are arguably the most important and frequently used instructional resource in massive open online courses (MOOCs). Recent research has explored learners’ perceptions and preferences regarding MOOC instructional videos. However, these studies are often limited to a small number of specific courses, and few grounded theory studies have been undertaken to investigate this topic. In the present study, a multiple-coder research methodology was adopted to analyze 4534 learner reviews of MOOCs in 14 categories. The study aimed to identify key characteristics associated with learners’ favorable perceptions of MOOC videos, types of supplemental or in-video resources learners perceive helpful to support MOOC video use, and video production features learners value. Results revealed that (a) “organized”, “detailed”, “comprehensible”, “interesting”, and “practical” were the top five important characteristics associated with learners’ favorable perceptions of MOOC videos; (b) learners perceived “presentation slides”, “reading materials”, “post-video assessments”, “embedded questions”, and “case studies” as helpful resources to support their utilization of MOOC videos; and (c) learners found “duration” a more salient production feature than “editing”, “resolution”, “subtitles”, “music”, or “voice”. The findings present implications for MOOC video design and foundations for future research avenues. Videos are arguably the most important learning resource in massive open online courses (MOOCs). As the primary instructional medium in MOOCs, videos play a critical role in explaining learner satisfaction with the instructional format . Text miInvestigating the key factors associated with users’ favorable perceptions of MOOC instructional videos can help obtain better-informed, richer insights into the design of instructional videos and related learning resources in MOOCs. Recent research has explored MOOC learners’ perceptions and preferences regarding the features of MOOC instructional videos . HoweverEmpirical studies undertaken to provide recommendations on the design of instructional videos in MOOCs can be divided into two broad categories. One research stream analyzes clickstream data to identify users’ interaction patterns while watching MOOC videos ,10. NotaThe second research stream employs self-report measures to understand learners’ perceptions and preferences regarding MOOC videos. For example, Mamgain et al.  designedAlthough MOOC learning typically revolves around watching instructional videos, it is also dependent on other instructional resources . ThroughScholars have conceptualized video production features as characteristics associated with planning and producing video content, such as showing the instructor’s face on the screen . ResearcVideo production features can be expensive or difficult to implement and are moderated by educational contexts. Unlike instructional video users in flipped and traditional classrooms , MOOC leIn contrast to capturing the opinions of small numbers of learners who took a specific MOOC, collecting reviews from multiple MOOCs or third-party aggregated MOOC platforms allows for coverage of broad perspectives. Existing MOOC studies have demonstrated the effectiveness of leveraging text-based reviews to obtain insights from large numbers of learners; data collected in this manner avoids the self-selection bias that can be present in data collected through interviews or surveys ,28,29. TThe study makes a valuable contribution to the literature on MOOC instructional videos—the most salient and prevalently used resource in MOOCs—because it collects and considers a large quantity of learner reviews across a broad spectrum of MOOCs, thus constituting a rich source of information for gaining insight into the design of MOOC instructional videos and related resources. The objectives of the current study are to reveal (1) key characteristics associated with learners’ favorable perceptions of MOOC videos, (2) types of instructional resources that learners perceive as helpful and supportive in their use of MOOC videos, and (3) video production features valued by MOOC learners.Previous research has shown that third-party platforms provide reliable sources of information about learners’ perspectives on the MOOCs they have participated in ,33. In tWe pre-processed the raw data in four stages to improve its quality prior to subjecting it to analysis . To thatWe adopted a grounded method to analyze the data. That is, we did not deliberately impose theoretical frameworks on the data corpus; rather, themes were guided by the research questions and allowed to inductively emerge during the iterative data-analysis process. For example, the following sentence “The videos were interpolated with questions and contributed to knowledge consolidation and reflection.” was coded as embedded questions, because it specifically emphasized interpolating instructional videos with questions.MAXQDA was employed to perform qualitative content analysis. Each sentence was treated as a unit of analysis. The multiple-coder research methodology we adopted in this study was adapted from Corsini et al. . Two teaNext, Teams A and B each used the refined codebook as a guide to code the entire sample independently. Inter-coder agreement analysis was subsequently conducted in MAXQDA to determine the level of agreement in coded segments between the two teams. The result (87%) indicated a high level of inter-coder agreement. To further improve research reliability, the teams resolved discrepancies by discussion and reinspection of data in face-to-face meetings. Consensus was considered reached when each coded segment was agreed upon by all members of both teams. Analysis results are displayed in the next section by research question. The original reviews were written in Chinese, and we translated partial excerpts into English. The translated excerpts were then checked by a professional English language editing service to ensure accurate usage.n = 1061), as reflected in the following three aspects. First, learners appreciate video content that explicates the logical relationships between key concepts: “The videos are carefully designed, and the knowledge points and logic are carefully clarified” (Accounting) and “The explanation is very logical…Not only are we told that this phenomenon happens to organisms, but how it was discovered and what it means biologically is also explained” (Molecular Biology). Second, learners appreciate it when the degree of difficulty in videos is elevated incrementally: “The video content transitions reasonably from easy to difficult” (Dance Sport) and “Video content is reasonably arranged, and a step-by-step approach from the easier to the more advanced parts is adopted” . Finally, learners applaud important and difficult information being made prominent in the videos. Two excerpts are illustrative: “The explanations in the video are very clear, and key parts are highlighted in red” (Animation Production) and “The instructor emphasizes key words through use of a heavy tone so that students can easily follow the instructor’s thought process and guidance” (Introduction to Public Finance).In analyzing learner’s reviews, “organized” was identified as an important theme (n = 962). It can be interpreted from two perspectives. On the one hand, learners appreciate it when videos provide detailed, thorough explanations, such as regarding financial knowledge: “The instructor explained investment and financial management information in detail”  and grammar knowledge: “Grammar issues are elaborated on by the instructor” (Chinese Grammar). On the other hand, learners value videos that provide detailed, step-by-step demonstrations, including, for example, software-operation procedures: “When it comes to [teaching] software, demonstrations of the specific operations makes learning easy” ; doing yoga: “The decomposition of movements is very precise and detailed” (Fitness Yoga); and dancing “The instructor demonstrated in great detail so that we could master the movements more accurately” .“Detailed” is another important theme identified (n = 443). Comprehensible video content is that which is readily comprehensible to the learner, as in the following excerpt: “The explanation in the video is easy to understand. It is not difficult for students with non-related majors to comprehend course content” . Learners attribute the comprehensibility of MOOC videos to various factors, including but not limited to the level of detail provided: “The videos elaborate on how to dribble the ball, and they demonstrate the corresponding action for different dribbling methods so that people can easily understand” ; inclusion of case studies: “Selected cases are used to make the content easier to understand” ; and video segmentation: “Although some of the course content is difficult for me, it becomes easy to understand when broken down into short videos” (Research Methods for Teachers).“Comprehensible” is an important theme among learners (n = 321). Interesting videos are those that are fun to learn from. Some MOOC instructors use specific communication styles and techniques to generate interest: “Dr Guo is very humorous. Students are not only impressed by his clear logical thinking but also attracted by his ‘funny’ personality”  and “The narration is entertaining and humorous, just like a story being told” (Research Methods for Teachers). Other instructors generate interest by designing video content to capture students’ attention, such as by incorporating amusing stories and case studies. The following excerpts illustrate ways in which instructors make learning content interesting: “Each video starts with a lively and interesting short story” (Entrepreneurship Foundation); “Dr Chen discusses many interesting cases in the videos” (Law); “What impressed me most was the personification of the ‘curriculum’ in the form of resumes and interviews, which made dry knowledge vivid and interesting” .Learners identified “interesting” as another theme important to them (n = 303). Learners applaud MOOC videos that impart knowledge and skills applicable to their present and future work, life, and study. The following two excerpts illustrate this point: “I learned so much from watching the videos. I learned a lot about how to dress and that body shape and image play an important role in personal impressions and job interviews” (Image Building) and “The examples given in the video reflect what we often come across, and they have helped me solve many problems” (Research Methods for Teachers). Similarly, learners like video explanations and demonstrations that stay close to life: “The content sticks very close to the daily work of the front-line teacher, and the sitcoms in the videos are representative” (Communication Arts for Teachers); “The examples given are relatively contemporary and are similar to everyday life” (Introduction to Public Finance).“Practical” was an additional important theme identified (n = 456). Learners generally appreciate being provided with slides of the presentations made in the videos: “It is super helpful that the videos are accompanied by the slides” (Botany). Learners found slides helpful when they summarize key information: “The slides are very well organized and summarize the content of the video succinctly” (Entrepreneurship Foundation); provide additional explanations: “The slides accompanying the videos helped me learn better, because they explained the course content in greater detail” ; and allow them to revisit the knowledge: “The slides accompanying the videos can help us better review and understand the lesson” (Civil Procedure).“Slides” are the most discussed resource supporting videos used in MOOCs (n = 363). Typical reading materials include downloadable files and rich texts such as open textbooks, book chapters, and articles. Learners believe that reading materials are useful when they expand horizons: “The best part is that numerous expanded readings accompany the videos, which really broadens your mind” (Pediatrics of Chinese Medicine) and consolidate knowledge: “The MOOC is taught not only in the popular video format but also in text format, which helps me consolidate the knowledge explained by the professor” (Emotion Management).Additionally, “readings” are perceived as helpful resources to support video-based learning (n = 360) and “embedded questions” (n = 38) as helpful. The former refers to computer-graded quizzes and peer assessments learners are required to complete after watching the videos. The latter are multiple-choice and true-or-false questions embedded in the videos. Learners maintain that post-video assessments are helpful because they allow them to assess their learning: “Each chapter contains a quiz, which is conducive to checking our understanding of the lectures” (Accounting) and consolidate knowledge: “The peer-graded assignment gave us opportunities to learn from others’ strong points to compensate for our own deficiencies” (Dance Sport). Learners also perceive “post-video assessments” ; learning assessment: “Inserting quizzes in the lectures worked well, because the questions tested the effectiveness of my learning” (Law); and knowledge consolidation: “The questions embedded in the videos helped me recapitulate what I learned” (Marketing).n = 180). Cases are either presented at the beginning of the videos or analyzed throughout. Learners believe that the inclusion of cases is helpful because cases make course content easier to understand: “When the instructor explains a theory that is strange to us, he cites easily understandable cases from life, which is really helpful for understanding the lesson”  and because they generate interest in learning: “Interesting cases are interspersed between relatively boring professional theories to make obscure knowledge come alive” (Law).Learners point out that “cases”, such as case studies and specific examples, are helpful in-video resources (n = 313). Learners generally prefer short videos because the brevity allows the focal points to stand out: “The videos are limited to 5 to 10 min, but each moment of content is important” (Pediatric Dentistry); “Sometimes a video covers only a single knowledge point, which makes that point easy to understand” (New Media Theories and Concepts). Moreover, learners note that short videos are helpful in reducing mental fatigue: “The videos are of moderate length and do not cause fatigue”  and maintaining attention: “Each video is short, allowing me to stay focused for a long time” (Effective Teaching). Learners also felt that short videos promoted flexible learning: “The duration of each video is not long, so I can make full use of my fragmented time” (Anatomy).Duration is the most discussed video-production feature (n = 106), high resolution (n = 86), subtitles (n = 51), appropriate music (n = 33), and clear voice (n = 20) in MOOC videos. Specifically, learners appreciate videos that are professionally edited: “Speaking of video production, the videos are beautifully edited, and the colors and font sizes are just right” . They also prefer high-resolution videos: “It must be mentioned that the videos in this course are so clear; even the standard definition is extremely clear” (Accounting). This is likely because high-resolution videos improve study experience: “High-definition videos offered better viewing experience” (Logistics) and facilitate practice of the procedural knowledge being taught: “The video is clear, making it easy to imitate the steps” (Learn the Piano from Scratch). Apart from duration, learners value quality editing  and organization of thought: “Some videos have subtitles to clarify the thoughts expressed” (Exercise Physiology).Finally, learners appreciate videos that feature appropriate background music: “The soundtrack complemented the instructor’s explanation really well” (Wood and Human Civilization) and instructors with clear voices: “The instructor’s voice is clear, so it’s very comfortable to watch the videos” (Software Engineering).The key findings of this study are summarized in This study’s finding that “interesting” is an important theme associated with MOOC videos corresponds with Peng and Jiang’s  observatThis study’s identification of “practical” as an important theme is consistent with recent research showing that MOOC learners emphasize the importance of acquiring knowledge that can be readily transferred to real-life contexts . MainstrThis study found that “slides”, “readings”, “post-video assessments”, “embedded questions”, and inclusion of “cases” were among supplemental and in-video resources that users cite as useful in augmenting their experience with MOOC videos. Past research has shown that course resources play a major role in facilitating MOOC engagement  and expeThis study revealed that embedded questions were perceived as helpful by MOOC learners to diagnose learning, consolidate knowledge, and actively process video content. Previous empirical studies showed that interpolating instructional videos with pop-up quizzes and open-ended questions led to improved academic performance when conducted in the context of credit-bearing online courses , flippedThis study found that compared to “editing”, “resolution”, “subtitle”, “music”, and “voice”, “duration” is the most important production feature to consider when designing instructional videos for MOOCs. MOOC learners often make it less than halfway through videos longer than 9 min . The freAlthough potentially important video production features have been identified, we maintain that the effectiveness of these features should be determined in future MOOC research. The effectiveness of video production features may be moderated by environmental factors , such asThe current study provides several implications for practitioners to design instructional videos to support student learning. First, MOOC videos should be “organized”, “detailed”, “comprehensible”, “interesting”, and “practical”. Instructors are advised to present logical relationships between key concepts, incrementally increase difficulty, and make important and difficult information prominent in videos. Videos should provide thorough explanations for declarative knowledge and step-by-step demonstrations for procedural knowledge. It is advisable to use strategies such as providing extensive details and utilizing case studies to improve learners’ comprehension of video content. Additionally, instructors should consider incorporating design strategies that facilitate emotional learning processes and the acquisition of practical knowledge and skills.Second, MOOC videos should be augmented with supplemental instructional resources, particularly “presentation slides”, “reading materials”, “post-video assessments”, and in-video resources, in particular, “in-video questions” and “case studies”. Instructors could design presentation slides to summarize key information, provide further explanations, and encourage learners to revisit videos to review knowledge. They could provide reading materials that expand horizons and foster knowledge consolidation. Meanwhile, instructors are recommended to design post-video questions and videos with embedded questions to help learners evaluate their learning, consolidate knowledge, and actively process video content. Moreover, instructors are advised to incorporate case studies and examples in videos to facilitate understanding and generate learning interest.Third, designing shorter videos for MOOCs to reduce learning fatigue and make important information stand out is pivotal. However, simply capturing a video lecture and dividing it into multiple shorter videos is not advisable . Rather,This study has several limitations. First, the key characteristics that emerged from the analysis of text-based reviews comprised frequently occurring categories in reviews. While frequency of occurrence is one way to identify salient characteristics, we acknowledge alternative means of conceptualization. Future research could define key characteristics differently or adopt automatic text-mining approaches to investigate this topic ,78.Second, we did not control for personal or course characteristics when processing the learner review data. As the course reviewers’ demographic characteristics were unknown, we were unable to analyze the association between learner perceptions of MOOC videos and learner characteristics. Future research is necessary to investigate how MOOC videos and relevant instructional resources can be designed to better cater to learners with certain characteristics or special needs, such as those who use assistive technologies or declare a disability . In addiThird, the text-based reviews examined in this study were collected from a Chinese MOOC platform only. These reviews may be influenced by users’ unique national and cultural attributes, and the findings may not be generalizable to the entire population of MOOC participants. Future research should analyze reviews collected from learners in MOOCs outside of China to provide a wider global perspective.Instructional videos have become ubiquitous in MOOCs and are integral to teaching and learning in MOOCs. However, they should not be viewed simply as a panacea for enhancing educational performance; suboptimal use of videos can waste students’ time and inflate their judgment of learning . To inveThe main contributions of this research consist of three key findings and recommendations to be adopted in the design of MOOC instructional videos. First, we discovered that learners favor MOOC videos they perceive as organized, detailed, comprehensible, interesting, and practical. Accordingly, we advise that MOOC videos be designed to characterize the following five attributes: “organized”, “detailed”, “comprehensible”, “interesting”, and “practical”. Second, we also discovered that learners perceive presentation slides, reading materials, post-video assessments, embedded questions, and case studies as helpful to support their use of MOOC videos. Thus, we suggest that MOOC videos should be augmented with supplemental instructional resources, particularly “presentation slides”, “reading materials”, “post-video assessments”, and in-video resources, in particular “in-video questions” and “case studies”. Third, we observed that duration was a more important production feature for instructors to consider than editing, resolution, subtitles, music, or voice when designing MOOC videos. Thus, we recommend that MOOC practitioners design shorter videos for MOOCs to reduce learning fatigue and highlight important information. We hope that the results of the study provide valuable insights into the design of instructional videos and resources to advance MOOC teaching and learning practices.


The formulation displayed rapid gelation in less than a minute on exposure to 0.1 N hydrochloric acid (pH 1.2) and remained afloat for more than 24 h. The formulations were capable of gradually releasing more than 80% of HCA load over 8 h, depending on the composition. The resulting gels exhibited high values of gel strength by texture analysis, suggesting they would offer resistance to breakdown under the action of stomach content movement. The optimized formulation loaded garcinia extract significantly reduced lipid accumulation in 3T3-L1 adipocyte cells and displayed moderate anti-inflammatory activity by inhibiting the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 macrophage cells. These findings demonstrate that oral in situ gelling liquid formulations based on sodium alginate and HPMC K100 offer much potential for sustained delivery of HCA and other anti-obesity compounds.Novel in situ gelling liquid formulations incorporating garcinia extract were developed to achieve prolonged delivery of hydroxycitric acid (HCA), an active compound displaying anti-obesity function, following oral administration. The optimized formulation was composed of sodium alginate (1.5%  Overweight and obesity are among the most prevalent risk factors associated with a number of acute and chronic health problems including type II diabetes, hypertension, osteoarthritis, cancer, and cardiovascular disease. Obesity in turn is linked to various causes, including a person’s genotype, a sedentary lifestyle, and excessive food consumption ,2. At prGarcinia atroviridis Griff. Ex T. Anderson also known as garcinia, Malabar tamarind, belongs to the plant family of Clusiaceae and is distributed mainly in Southeast Asia .The inhibitory effect of HCA on nitric oxide (NO) production in murine macrophages like RAW 264.7 cells was evaluated using a modification of the method reported by Kaewkroek et al., 2019 . Cells w reagent . The optt-test or one-way analysis of variance (ANOVA). p < 0.05 and p < 0.01 were regarded as statistically significant.Data were presented as mean ± standard deviation (mean ± S.D.). The data were processed by Student’s


Caenorhabditis elegans. We found a finely regulated antagonistic balance between PVD-expressed KPC-1 and the epidermally expressed putative cell adhesion molecule MNR-1 (Menorin). Genetically, partial loss of mnr-1 suppressed partial loss of kpc-1, and both loss of kpc-1 and transgenic overexpression of mnr-1 resulted in indistinguishable phenotypes in PVD dendrites. This balance regulated cell-surface localization of the DMA-1 leucine-rich transmembrane receptor in PVD neurons. Lastly, kpc-1 mutants showed increased amounts of MNR-1 and decreased amounts of muscle-derived LECT-2 (Chondromodulin II), which is also part of the Menorin adhesion complex. These observations suggest that KPC-1 in PVD neurons directly or indirectly controls the abundance of proteins of the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback from the dendrite to the instructive cues of surrounding tissues.During neural development, cellular adhesion is crucial for interactions among and between neurons and surrounding tissues. This function is mediated by conserved cell adhesion molecules, which are tightly regulated to allow for coordinated neuronal outgrowth. Here, we show that the proprotein convertase KPC-1  regulates the Menorin adhesion complex during development of PVD dendritic arbors in  Summary: The neuronal furin-like proprotein convertase KPC-1 in Caenorhabditis elegans requires catalytic activity to non-autonomously regulate the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback during dendrite development. Animal behavior relies on functional neuronal networks, which in turn rely on sensation of sensory stimuli and their coordinated processing. Defects in sensory processing of both auditory and mechanosensory modalities have been implicated with neuropsychiatric disease . The sinCaenorhabditis elegans are a well-established model for studying dendrite development . This allele changes a conserved arginine residue in close proximity to the catalytic histidine 262, and results in a significant increase in the number of secondary branches and defects in self-avoidance of tertiary branches  partial loss-of-function allele, suggesting that they likely affected the kpc-1 locus (kpc-1(R265Q). Here, we focus on the dz213 allele, which completely suppresses all kpc-1(R265Q) mutant phenotypes.Complete loss of KPC-1 function causes defects in dendrite branch extension, branch number and self-avoidance of tertiary dendrites in PVD neurons, likely owing to increased adhesion of the Menorin complex . To idenbranches B, Fig. 2branches . Of ten henotype . We alsohenotype  or will -1 locus . The remdz213 suppressor allele as a missense mutation in mnr-1 (kpc-1(R265Q) mutant animals (mnr-1(dz213) [hereafter referred to as mnr-1(L135F)] is a partial loss of function allele. First, mnr-1(L135F) failed to complement the putative null allele mnr-1(dz175), both with regard to the number of branches and the dendrite self-avoidance defects (L135F) were required for suppression of the kpc-1(R265Q) PVD phenotype, showing that mnr-1(L135F) is recessive (mnr-1 cDNA in the epidermis rescued the mnr-1(L135F) mutant, i.e. it reversed the suppression of the kpc-1(R265Q); mnr-1(L135F) double mutant, and overexpression of a mnr-1(L135F) mutant cDNA partially rescued mnr-1(dz175) null mutant animals (kpc-1 function because (1) the suppression is dosage sensitive (mnr-1(dz175) putative null allele could not suppress the kpc-1(R265Q) hypomorphic phenotype (mnr-1(L135F) mutant cDNA driven by an epidermal promoter completely rescued the mnr-1 null phenotype in kpc-1(R265Q); mnr-1(dz175) double-mutant animals (data not shown). Finally, the mnr-1(L135F) allele is temperature sensitive because mnr-1(L135F) mutant animals displayed PVD phenotypes at the non-permissive temperature of 25°C that were indistinguishable from the mnr-1 null phenotype, but no phenotype at all at the permissive temperature of 20°C (mnr-1(L135F) allele in kpc-1(R265Q); mnr-1(L135F) double mutants was lost at the non-permissive temperature compared with at the permissive temperature (mnr-1(L135F) is dosage sensitive and (2) that some MNR-1 function is required to suppress a partial loss of KPC-1 function. Thus, we conclude that mnr-1(L135F) is a recessive, partial-loss-of-function allele of mnr-1, which can suppress the defects of a partial-loss-of-function allele of kpc-1. Importantly, only reducing but not eliminating mnr-1 function can compensate for reduced kpc-1 function.Using whole-genome sequencing, mapping and transformation rescue, we identified the molecular lesion of the in mnr-1 . This miin mnr-1 A. The L1 animals B-D,F. Se defects E-G. Secoecessive D,E. Thir animals . Some MNensitive G; (2) thhenotype ; and (3) of 20°C . All supperature , also sukpc-1 partial-loss-of-function phenotype in the mnr-1(L135F) allele is dependent on some kpc-1 function. Existing kpc-1 alleles form an allelic series as follows: gk8=gk779937>dz185>gk333538>dz254, where gk8 represents the putative null phenotype with the strongest phenotype and dz254 represents the weakest phenotype (mnr-1(L135F) allele completely suppressed the weaker kpc-1 alleles (dz254 and gk333538), but only partially suppressed the intermediate kpc-1(dz185) allele (kpc-1(gk8) null allele  and the kpc-1(gk779937) (G263R) strong-loss-of-function allele were not suppressed by the mnr-1(L135F) allele (kpc-1 by loss of MNR-1 function is (1) sensitive to both the dosage of MNR-1 and KPC-1, and (2) requires residual function of both genes.We next asked whether, conversely, suppression of the henotype   allele . The put) allele . In concmnr-1 could have similar effects: adhesive forces exerted by overactivation of the Menorin complex from the epidermis could prevent dendrite extension. To test this hypothesis, we first expressed a fosmid containing mnr-1 under control of most, if not all, regulatory regions at low doses (1 ng/µl) in mnr-1(dz175) null mutants and found that it, as expected, completely rescued the PVD defects of mnr-1 mutant animals  resulted in a dramatic transformation of the PVD dendritic tree from a mnr-1-like phenotype to a kpc-1-like mutant phenotype (kpc-1 null mutants (kpc-1 function was completely lost [kpc-1(gk8)], dendrites became essentially trapped in the vicinity of the primary dendrite and self-avoidance defects could not be detected  by promoting endocytosis of the DMA-1 receptor . Our gen animals A,D. Inte alleles B,E. Morehenotype C. Specif mutants C,D,F. Nodetected C. Similapromoter C. Togethpromoter G.kpc-1 loss-of-function phenotype in PVD is the increased level of DMA-1 on the membrane. This has been suggested to result from diminished endocytosis of the DMA-1 receptor, based on colocalization in endocytic vesicles and co-immunoprecipitation experiments indicating that both KPC-1 and DMA-1 are part of a biochemical complex  null mutant animals carrying (1) a transgene that ectopically expresses MNR-1 in muscle cells, (2) a transgene that expresses a DMA-1::GFP fusion in PVD and (3) a transgene that expresses myristoylated mCherry in PVD. We calculated the GFP (DMA-1) to mCherry (membrane) fluorescence ratio in tertiary branches, normalized to that in secondary branches, and compared it with the ratio in a control strain expressing only the DMA-1::GFP fusion protein and myristoylated mCherry in PVD  rescued the defects ::sfGFP] expressed in PVD displayed protein expression levels and localization in PVD dendrites comparable with those of the analogous wild-type KPC-1::sfGFP reporter  accumulates in coelomocytes, a set of scavenger cells that uptake diffusible proteins from the pseudocoelom, raising the possibility that MNR-1 is shed from cell membranes or secreted . We ther animals . These o animals . In cont animals . We next mutants . Lastly,tterning   allele we isolated, is functionally important, and likely requires calcium for its catalytic activity activity , a findiactivity  but distactivity . Taken tkpc-1 mutants  by which KPC-1 convertase activity regulates dendrite morphogenesis in PVD neurons.Our results raise the possibility that MNR-1 is shed from the epidermal membrane, whereas other factors of the MNR-1 pathway for PVD dendrite patterning, such as the epidermal cell adhesion molecule SAX-7 or the DMA-1 receptor itself, appear to be unaffected by loss of KPC-1 . Unexpec mutants B. A posseviously . Adhesiveviously . Our geneviously : on one in cis, i.e. with both furin and reelin functioning in the same cell  was injected at 1 or 20 ng/μl into mnr-1(dz175); dzIs53 [Is(F49H12.4::mCherry)] animals with myo-2p::mCherry as an injection marker at 25 ng/μl and pBlueScript (Stratagene/Sigma-Aldrich) at 50 ng/μl. The same injection mix with 1 ng/μl of WRM618aD06 was injected into dzIs53 control animals to assess putative mnr-1 overexpression effects on PVD dendritic arbor morphology.A fosmid containing the dzIs49[Is(myo-3p::mnr-1)] integrated strain that expressed MNR-1 in the body wall muscle of a mnr-1 null mutant strain (rol-6(su1006). We used this strain to quantify DMA-1::GFP expression levels when MNR-1 was expressed in the muscle. To control for the presence of the rol-6(su1006) injection marker in dzIs49, we injected otherwise isogenic animals without the integrated transgene mis-expressing MNR-1 in muscle with 40 ng/μl of rol-6(su1006) and 60 ng/μl of pBlueScript. Transgenic animals and non-transgenic siblings were used as controls for fluorescence intensity quantification and the acquisition of comparative images in We previously generated the t strain . The injkpc-1 cDNA tagged at the C-terminus with sfGFP was cloned under control of a short version of the ser-2prom3 promoter (which we term ser-2p3s) or the rab-3p promoter, and injected at 5 ng/μl into dzIs53 [Is(F49F12.4p::mCherry)] animals either with myo-2p::mCherry or rol-6(su1006) as injection markers at 25 ng/µl together with pBlueScript at 50 ng/μl. A mutagenized version of the ser-2prom-3s::KPC-1::sfGFP plasmid carrying the H262A substitution in KPC-1 was used to assess rescue and protein expression of this catalytically dead form of KPC-1.kpc-1 containing the signal sequence of KPC-1 but lacking its prodomain (H34 to R146) was cloned downstream of the ser-2p3s promoter. Two catalytically dead versions of this preprocessed, prodomain-less construct were generated by site-directed mutagenesis, including H262A  and N363A  (cDNA of ertases) .mnr-1 plasmids doubly tagged with either N-terminal mCherry or tagRFP and C-terminal mNeonGreen were generated by digestion and ligation of individual N-terminal- and C-terminal-tagged mnr-1 (cDNA) constructs  animals at 1 ng/μl together with the unc-122p::tagBFP marker at 25 ng/μl, the F49H12.4::tagBFP marker at 25 ng/μl and pBlueScript at 50 ng/μl.nstructs . The findpy-7p::mnr-1 rescuing construct was injected directly into kpc-1(gk333538); mnr-1(dz213) double-mutant animals or used for site-directed mutagenesis to introduce the L135F substitution. The L135F mutant construct was injected into kpc-1(gk333538); mnr-1(dz175) double-mutant or kpc-1(gk333538) or mnr-1(dz175) single-mutant animals. All constructs were injected at a concentration of 5 ng/μl together with 25 ng/μl of myo-3p::GFP or myo-3p::tagRFP injection markers and 70 ng/μl of pBlueScript.A kpc-1 cDNA sequence under control of the ser2prom3s or the mec-7p promoters (kpc-1(gk8); dzIs53; hmIs4 animals in which the defective dendritic arbors of PVD and FLP were visible. Constructs were injected at a concentration of 5 ng/μl either with 25 ng/μl of myo-3p::tagRFP or ttx-3p::mCherry injection markers, and 70 ng/μl of pBlueScript DNA.Plasmids carrying the romoters  were indkpc-1(gk333538); dzIs53 animals were mutagenized with ethyl methane sulfonate and the progeny of cloned F1 animals was scored for suppression or enhancement of the kpc-1 hypomorphic dendrite branch defects  mutant animals displayed shorter 1° branches and self-avoidance defects but not an increased number of 2° branches, compared with those of wild-type control animals  deletion  comprised 100 μm of the primary branch anterior to the PVD cell body. Morphometric analyses were conducted using the NeuronJ plugin of the FIJI software and branches were defined as follows: 2° dendritic branches as any neurite branching out of the primary dendrite in the ROI; 3° branches as any neurite coming from the tip of a 2° branch and located in close proximity to the sub-lateral nerve cords; and 4° dendritic branches as those originating from 3° dendritic branches and extending to the dorsal or ventral nerve tracts. The self-avoidance index was defined as the ratio of the number of gaps between adjacent 3° branches divided by the number of candelabras within the ROI.3° branch regions in the same candelabra/baobab and the corresponding 2° branch were used for DMA-1::GFP quantification in FIJI. Three different regions were quantified per candelabra or baobab , whereas one region was quantified for the corresponding 2° branch. Normalization was performed first by dividing the fluorescence signal in 3° branches by the fluorescence signal in the corresponding 2° branches  and then by dividing this ratio by the same ratio obtained for the same anatomical structures in the red channel . Red and green integrated density (ID) values were recorded for each ROI, avoiding regions with obvious green DMA-1::GFP puncta. For the mean background intensity value , a circle with the same diameter (3 μm) was drawn adjacent to the ROI but outside of the branches and avoiding gut autofluorescence. The exposure time used for the red and green channels was 100 ms for both strains (EB3585 and EB3595); overexposed animals were excluded from the analysis and the fluorescent intensity ratio (FIR) was calculated using the following formula:The same procedure was performed for DMA-1::GFP fluorescence quantification in epistatic analyses, with few modifications: (1) three ROIs per 2° branch were quantified and (2) the arbitrary fluorescence intensity ratio was calculated as follows:mCherry::mnr-1::mNeonGreen construct (EB3599 and EB3600). The exposure time used for the red, green and blue channels was 100 ms, 100 ms and 50 ms, respectively, for strains carrying the tagRFP::mnr-1::mNeonGreen construct (EB3658 and EB3682). The fluorescence ratio was calculated using the following formula:L1 animals were synchronized by starvation overnight after egg preparation and, after 48 h on food, L4 animals were imaged. For quantification purposes, a single optical section in the mid-equatorial plane of the pair of coelomocytes located in the medial section of the animal was taken using a 40×/1.4 objective on a Zeiss AxioImager Z1 Apotome. The coelomocytes were encircled using the freehand selection tool in Fiji (ROI). Blue and red (or green) ID values were recorded for the exact same ROI. For the MBV value, a small circle, 3 μm in diameter, was drawn adjacent to the coelomocyte ROIs but avoiding gut autofluorescence. The exposure time used for the red, green and blue channels was 50 ms for strains carrying the kpc-1 was created using the I-TASSER server (5JXG). The chosen model had a confidence score of −0.48 and an estimated TM-score  of 0.65. The pro-domain was removed from the sequence prior to simulation. The editing of the structure was performed using Chimera  and edited using Chimera. The pro-domain extends from T61 to Q152  and collected in a 15 ml Falcon tube. Following centrifugation at 1000 g for 5 min, the worm pellet was resuspended in HBS at 4× the volume of the pellet and 10% SDS at 1× the volume of the pellet. The suspension was sonicated with a probe sonicator at 40% power for 1 min with a 5 s on/5 s off cycle and centrifuged at 20,000 g for 10 min. The supernatant was combined with 4× SDS loading buffer and incubated at 60°C for 15 min, before loading onto gels for SDS-PAGE . Standard SDS-PAGE and wet-transfer western blotting were followed with 5% milk used for blocking. Antibodies for western blots were used at the following concentrations: for anti-FLAG blots, 1:800 anti-Flag  and 1:5000 HRP-conjugated anti-mouse IgG ; for anti-GFP blots: 1:500 anti-GFP  and 1:5000 HRP-conjugated anti-mouse IgG ; for anti-HA blots, 1:500 anti-HA  and 1:10000 HRP-conjugated anti-rat IgG ; and 1:5000 anti-α tubulin .For immunoprecipitation, five full plates of DMA-1::GFP transgenic worms were washed in RIPA buffer pH 7.0  and lysed for 15 min in a Biorupter water bath, as previously described for whole-worm protein extraction . Post lymnr-1 fosmid overexpression experiments were calculated by comparing transgenic and non-transgenic siblings using an unpaired, two-tailed t-test. DMA-1::GFP fluorescence ratio differences for epistasis analysis and for mis-expression of mnr-1 experiments were calculated using a one-way ANOVA with Sidak's correction for multiple comparisons. For proportions, statistical significance was calculated using the z-test with Bonferroni correction for multiple comparisons where applicable. For comparisons of coelomocyte fluorescence intensity ratios, a non-parametric Mann–Whitney test was performed. All tests were performed using the Prism 7 Statistical Software suite from (GraphPad). Statistical significance is indicated as ns, not significant; *P≤0.05; **P≤0.01; ***P≤0.001; and ****P≤0.0001.Statistical tests were applied as described in each legend. Statistical significance of the number of 2° branches, 4° branches or self-avoidance index were calculated with one-way ANOVA using Sidak's correction for multiple comparisons. Statistical differences in self-avoidance and 4° branch number in Click here for additional data file.10.1242/develop.201208_sup1Supplementary informationClick here for additional data file.


Plasmodium malariae  infection diagnosis remains unknown.Malaria is a worldwide infectious disease. For countries that have achieved malaria elimination, the prevention of re-establishment due to infections in returned travellers has become important. The accurate and timely diagnosis of malaria is the key in preventing re-establishment, and malaria rapid diagnostic tests (RDTs) are frequently used due to their convenience. However, the RDT performance in P. malariae cases from 2013 to 2020 in Jiangsu Province and evaluated the sensitivity of four parasite enzyme lactate dehydrogenase (pLDH)-targeting RDTs  and one aldolase-targeting RDT(BinaxNOW) for P. malariae detection. Furthermore, influential factors were investigated, including parasitaemia load, pLDH concentration and target gene polymorphisms.This study analysed epidemiological features and diagnosis patterns of imported P. malariae infection was 3 days, which was longer than that with Plasmodium falciparum  infection. The RDTs had a low detection rate  among P. malariae cases. All tested RDT brands had poor performance in P. malariae detection. All the brands except the worst-performing SD BIOLINE, achieved 75% sensitivity only when the parasite density was higher than 5000 parasites/μL. Both pLDH and aldolase showed relatively conserved and low gene polymorphism rates.The median duration from symptom onset to diagnosis among patients with P. malariae cases was delayed. The RDTs had poor performance in P. malariae diagnosis and may threaten the prevention of malaria re-establishment from returned travellers. The improved RDTs or nucleic acid tests for P. malariae cases are urgently needed for the detection of imported cases in the future.The diagnosis of imported  Plasmodium malariae is frequently coendemic with Plasmodium falciparum in sub-Saharan Africa, South America, Southeast Asia and the western Pacific [P. malariae is different from that of P. falciparum. Plasmodium malariae can establish a large parasite reservoir in asymptomatic carriers with low parasitaemia. Although P. malariae infection is relatively mild compared with P. falciparum infection, it can develop into chronic nephrotic syndrome, which has a high rate of mortality [Each year, millions of malaria cases occur worldwide, especially in tropical and subtropical regions . Plasmod Pacific . The traortality .With the development of international communication and cooperation, the increasing movement of the population between countries leads to a number of travel-related infections, and malaria is one of the main infections diagnosed in African countries and workers . For couPlasmodium species. Reported RDT sensitivity is generally good for P. falciparum, but only moderate for Plasmodium vivax (66.0–88.0%) [P. malariae and Plasmodium ovale  using RDTs is less accurate and highly heterogeneous, mainly because of limited sample numbers [P.ovale, even when the parasitaemia load was larger than 500 parasites/µL [P. malariae, there are even fewer related studies and tested samples.Microscopy and rapid diagnostic tests (RDTs) are recommended by the World Health Organization (WHO) for confirmation of diagnosis in suspected malaria patients . Due to 0–88.0%) . Detectisites/µL . For P. P. malariae cases is a major concern. In this study, the characteristics of imported P. malariae cases were analysed and the sensitivity of five RDTs in detecting P. malariae to identify the ideal diagnostic methods was evaluated, which can be applied in the POR of malaria. Moreover, the factors that influenced the results of RDTs in the detection of P. malariae were also investigated.Considering past malaria epidemic patterns, whether RDTs are useful in diagnosing imported P. malariae cases in Jiangsu Province were collected from the China Information System for Disease Control and Prevention (CISDCP).Imported malaria cases in China and Jiangsu Province from 2013 to 2020 were analysed. Information on national imported malaria cases was collected from previously published papers. Epidemiological data of imported The age, sex and origin country distributions of the reported imported malaria cases were collected and analysed, and the duration between patients’ return to China to symptom onset and symptom onset to first medical facility visit were analysed.Clinic attendees with fever symptoms were tested for malaria by either microscopy examination or a RDT; both methods were used in medical facilities at the county and above levels. Positive results were reported via the CISDCP within 24 h, and 5 ml venous whole blood was collected for further confirmation. The Center for Disease Control and Prevention in the county where the case was reported rechecked the slides sent by the medical facilities or analysed the collected venous blood sample. Finally, all the whole blood samples were sent to the provincial laboratory within 2 weeks, where all the samples underwent both microscopic examination and nucleic acid-based molecular testing (PCR) . The samP. malariae-positive blood samples, confirmed both by microscopy and a nested PCR assay, were included in the study; mixed infection samples were excluded [The patients included international travellers and those who worked abroad from 2013 to 2017. Blood samples were collected from malaria patients at local designated hospitals and health centres in Jiangsu Province. Five millilitres of venous blood was collected by venepuncture into an EDTA tube. The collected blood samples were preserved at 4 °C and transported to a provincial malaria diagnostic reference laboratory for reconfirmation. Only excluded .The peripheral blood of patients was prepared as thick and thin films. Both smears were stained with 3% Giemsa for 30 min at room temperature. All slides were read under100x oil immersion. Thick films were used for counting parasites. Leucocytes, regarded as 8000/µL, and parasites were used to determine the parasite density . All of Plasmodium spp. BinaxNOW targets Pan-aldolase to detect Plasmodium spp. The stored frozen samples were used. Each blood sample was tested by all the five RDTs, with some exceptions due to limited sample volumes. All of the detection procedures followed the manufacturers’ instructions.Five RDTs were evaluated: Wondfo Diagnostic Kit for Malaria (Pf/Pan) , SD BIOLINE Malaria Ag Pf/Pan (Standard Diagnostic Inc. Product code 05FK60 lot 05EDC028A), CareStart Malaria Pf/PAN (HRP2/pLDH) Ag Combo RDT ,BioPerfectus diagnostic kit for malaria , and BinaxNOW . The first four brands target Pan-LDH to detect Plasmodium spp. Then, a labelled anti-pLDH antibody is bound to the complex, allowing a chromogenic reaction. As soon as the stopping solution is added, the colour intensity is proportional to the concentration of pLDH in the sample. Forty-five P. malariae samples were tested in this study. All procedures followed the kits’ instructions. The results were read with a spectrophotometer at 450 nm/620 nm.A quantimal pLDH CELISA kit  was used to quantify the density of pLDH in malarial blood samples. The detection principal is sandwich ELISA. The kit uses anti-pLDH capture antibody precoated microwells to bind pLDH in the samples to all P. malariae aldolase gene was amplified using primers ALf (5′-CAGGCATCAAGCGCAGACTA-3′) and ALr (5′-TAAAGCCCATGGGTGAGGTC-3′) [P. malariae LDH gene was amplified using primers LDHf (5'-ACTTTACAGCCGCCCATTCC-3') and LDHr (5′-CCTTCATTCTCTTCGTTTCAGCA-3′) [Genomic DNA was extracted from 200 µL blood samples with a QIAmp Blood Mini Kit (QIAGEN) according to the manufacturer’s instructions. The GGTC-3′) , while tAGCA-3′) . Conventp value < 0.05 were considered as significant.All the results were analysed using SPSS 19.0 and GraphPad Prism 8.3.0. Mann–Whitney test was used to analyse the variance of epidemiological data. The sensitivity of each brand as well as the sensitivity of each brand at different parasite density levels were assessed. The sensitivities of the 4 LDH-targeting brands at different pLDH concentration levels were calculated. Uncertainty was interpreted with 95% confidence intervals (CIs). Categorical variables were determined by Chi-squared tests. Fisher’s exact correction was applied when the expected frequency in the cell was 5 or less. Pearson’s correlation analysis was used to analyse correlation between parasite density and pLDH OD level. All the P. malariae cases at the national level in China and 85 cases in Jiangsu Province were diagnosed. During the 8-year period, there was a general increase in the number of P. malariae cases at the national level, from 51 cases in 2013 to 97 cases in 2019. Due to the decreased travel caused by coronavirus disease 2019 (COVID-19), the number of imported P. malariae cases in China decreased to 22. Among these, there were more than 10 imported P. malariae cases nearly every year in Jiangsu Province from 2013 to 2019  and aged 41–50 years . All of the cases originated from Africa, among which Angola accounted for the most cases , followed by Equatorial Guinea  and Nigeria  , and the median for P. malariae was 3 days , which was longer than 2 days (IQR: 1–4) for P. falciparum  were misdiagnosed as negative via RDTs  ) at the county level which exhibited a relatively low detection rate.All 69 P. malariae monoinfection cases from 2013 to 2017 were included in the study. The performances of the five RDTs  were compared with regard to P. malariae detection. Each sample was tested using all five brands, with some exceptions due to limited sample volumes. The results indicated that CareStart test had the highest sensitivity, at 72.7% (95% CI 59.0–86.4%), while the SD BIOLINE test had the lowest sensitivity, at 13.3% (95% CI 3.0–23.7%) . The SD BIOLINE test had only 41.7% sensitivity, even at parasite densities above 5001 parasites/μL (Table P < 0.05). All four RDTs exhibited poor performance (sensitivities from 33.3 to 66.7%) when parasite density was low (≤ 1000 parasites/μL), while the SD BIOLINE test could not detect any P. malariae when the parasite density was lower than 1000 parasites/μL . Sensitivity was calculated based on the performance of each brand in each group. The Wondfo, CareStart, BinaxNOW and BioPerfectus tests had sensitivities of 75%, 83.3%, 83.3% and 75%, respectively, when parasite densities were higher than 5000 parasites/µL. The categorical variable parasite densities were analysed and showed a result of no significance .The pLDH concentrations in the samples were quantified through ELISA test. The results were presented as optical density (OD) values. To correlate pLDH concentrations with the sensitivities of the four pLDH-based RDTs, 45 samples were divided into three groups  according to their pLDH concentrations (Table P < 0.0001), which represents a moderate correlation between the two factors.The correlation between pLDH concentration and parasite density was evaluated to assess whether the pLDH concentrations in samples were associated with the samples’ parasite densities Fig. . The r vP. malariae LDH gene from 39 samples yielded 982 base pairs, and the P. malariae aldolase fragments from 39 samples yielded 912 base pairs. No nucleotide substitutions were observed in the LDH gene of P. malariae compared to the reference sequence (NCBI Reference Sequence: XM_029006607.1). Three nucleotide substitutions were observed in the aldolase gene of P. malariae compared to the reference sequence (NCBI Reference Sequence: XM_029006963.1). Among the substitutions, 2 were synonymous mutations, and 1 was a nonsynonymous substitute with T681A in 4 samples.pLDH and aldolase sequences from samples were obtained, and the results were analysed with MEGAX. Amplification failed in some low-parasitaemia samples. However, the PCR products of the P. malariae cases have been imported every year and have shown an increasing trend in recent years. The imported P. malariae cases all originated from Africa and mainly occurred in males, which coincided with previous findings that most of the imported malaria cases in China occurred in workers returning from Africa and other malaria-endemic areas [P. malariae cases had longer durations from symptom onset to diagnosis than falciparum cases. These findings may attract attention because the longer the time for patients to be diagnosed after symptom onset is, the higher the risk of severe symptoms is. It also increases the risk of introducing malaria locally, posing a challenge in the prevention of malaria re-establishment.There have been thousands of imported malaria cases annually since 2013 in China; ic areas . These PP. malariae cases, the false-negative rate of RDTs was 43.5% at the local level. All five RDTs we tested showed poor performance for P. malariae detection, with relatively low sensitivity. To date, the WHO has not recommended criteria for P. malariae testing. Moreover, by using the same sensitivity thresholds for determining whether a RDT adequate detects P. falciparum and P. vivax, all the tested RDT sensitivities in this study for P. malariae are inadequate.The WHO recommends that all suspected malaria cases receive parasitological confirmation before drugs administered. Microscopic examination used to be the most popular detection method for malaria parasites. In recent years, RDTs have been recommended by the WHO to assist in the rapid and accurate diagnosis of malaria and have been gradually increasingly applied, especially in the preliminary screening of suspected cases. It could be predicted that in non-endemic areas, which may face challenges in maintaining microscopic examination capability, RDTs will be used more widely and even replace microscopic examinations at the local level. However, according to the 69 analysed P. malariae blood samples, and similar results were also observed in P. vivax and P. ovale samples and in the rodent model for malaria [P. malariae infection patients might have contained parasites at different developmental stages, the pLDH concentration instability during the metabolic process in Plasmodium parasites may have been the reason for the moderate correlation [P. malariae detection.In this study, the sensitivity of Wondfo, CareStart, BinaxNOW and BioPerfectus tests were not statistically increased with an increasing parasite density. All of the tests reached more than 70% once the parasite density was more than 5001 parasites/µL, with the exception of the SD BIOLINE test. The four  Pan-LDH-targeting tests had a different sensitivity with an increasing concentration of pLDH. The sensitivities of all the tests except the SD BIOLINE test reached 100% once the concentration of pLDH was more than 1.50 OD. However, a higher parasitaemia load was not correlated with a higher pLDH concentration; only a moderate correlation was observed between the pLDH concentration and parasite density in the  malaria , 22, 23.relation , 25. TheP. malariae-endemic areas with thousands of cases annually, Jiangsu Province included [P. malariae cases among travellers without early diagnosis and appropriate treatment influence the risk of re-establishment, which has been observed in many other countries where malaria has been eliminated for many years such as Armenia [P. malariae usually causes less severe disease in humans than other forms of malaria, it can still cause chronic nephrotic syndrome, which may lead to death [P. malariae cases efficiently and accurately, which increases the risk of severe symptoms and introduced malaria, posing a challenge to the malaria re-establishment prevention.China was officially awarded the national malaria-free certification from the WHO on June 30, 2021. However, the risk of the re-establishment of malaria from imported cases remains a concern due to increasing international trade and global exchanges . Historiincluded . Althoug Armenia . In addito death , 30. UnfP. malariae endemic situation, inefficient and inaccurate RDTs may increase the missed or delayed diagnosis on P. malariae, which leads to the adverse clinic consequences even the secondary transmission. Therefore, the diagnosis of P. malariae cases must attract more attention, and more advanced RDT products or other methods, such as nucleic acid-based molecular tests, should be developed and adopted to overcome this problem.Furthermore, for countries in Africa, southeast Asia and other regions with


Climate change and increasing global temperatures may favor the expansion of areas with C4 grasses, previously occupied by C3 plants.Warm-season  perennial grasses are widely spread and dominant in tropical and subtropical regions, between 30oN and 30oS latitude, and it is perceived that more than two-thirds of all grasses in these regions are C4. The most extensive natural C4 biomes are the various savannas of the tropics, subtropics, and warm temperate areas that range in C4 grass cover from 20% to nearly pure C4 grassland  may favor the dominance of C4 species in different ecosystems, therefore, management practices may have to be adjusted accordingly. 4 grasslands through the displacement of C3 vegetation during the Late Miocene and Pliocene. Corroborating with those findings, 3 to C4 species in the desert steppe in China, as indicated by the decrease in C3/C4 ratio due to warming temperatures over a 6-yr period. The expansion of C4 grasses in these areas may be detrimental to biodiversity, but it may not be avoidable due to the decline of the native vegetation and aggressive propagation mechanisms of C4 grasses.Temperature has been the most significant factor dictating the presence or persistence of C4 carbon fixation pathway, identified by 4 grasses range from wetlands to deserts and some of the least productive to the most productive ecosystems on the earth  in North America, kangaroograss  Stapf.) in northern Australia, thatchingrass (Hyparrhenia spp.) in South Africa, and bluestem (Andropogon spp.) in Brazil, which tend to be malate forming  enzyme variants. These locations are characterized by relatively high humidity and these grasses are usually managed at low-input levels  has been a variable of interest, primarily in extensive grazing systems with low N inputs. The NUE may be due to variations in the efficiency of the photosynthetic apparatus, such as enzyme kinetics.Besides the importance of the climatic conditions to different groups, soil fertility, and management conditions may interfere with the adaptability of CPanicum and found that at high levels of N availability the NAD-Malic species had higher shoot N concentrations than the NADP-Malic species. The NADP-Malic species may have a greater photosynthesis rate than NAD-Malic species at a given leaf N concentration. However, this trial compared subtypes within the same species and there may be larger inter-species variations.In general, it is expected that NADP-Malic plants have low late season forage N concentration (<5.0 g/kg), while aspartate forming  plants usually have greater tissue N concentrations (10–20 g/kg) . LeCain The NAD-Malic plants may have relatively greater N requirement than NADP-Malic, primarily due to greater enzymatic steps and amino acids . SuperioRecently, there has been identified genes related to NUE in crops. Genes that code for nitrate and ammonium transporters that assimilate N from the soil and genes that synthesize N compounds such as glutamine synthetase, which produces the amino acid glutamine (used to transport N through the plant) are examples of genes that have been recently evaluated to increase NUE in crops . The ideNative and planted grasslands are frequently found in soils with limited production capability, many times in highly weathered, acid, and less fertile soils. Soil microorganisms, pH, structure, texture, organic matter, water, and many other characteristics influence forage growth, however, soil fertility is one of the most influential characteristics in forage production. The combination of low soil nutrient levels, efficient nutrient extraction capability, and high total yield potential create conditions for significant yield responses when grasslands are fertilized .Cynodon spp.) fertilized from 0 to 160 mg N/kg and there was a significant correlation between tiller number and DM accumulation. Moreover, N fertilization also increases the number of leaves per tiller and leaf appearance rate. Panicum maximum Jacq.). Leaf appearance rate ranged from 0 to 0.14 leaves/d and leaf elongation from 25 to 60 mm/d with N fertilization levels from 0 to 200 mg/dm3. Santos et al. (2009) observed that leaf elongation rate increased from 15.4 to 46.8 mm/d in ‘Marandu’ palisadegrass [Brachiaria brizantha (Hochst.) ex A. Rich] with N fertilization levels of 0 and 100 kg/ha, respectively.Nitrogen is the most limiting plant nutrient for growth of grasses and yield and increasing crude protein (CP) responses have been observed with different species . Nitroge4 grass regrowth and persistence; therefore, a combination of fertilization and adequate levels of defoliation frequency and intensity should be imposed to promote persistence of C4 grasslands. It has been observed that N fertilization may not increase root-rhizome mass in warm-season forages ; however4 grasslands are managed extensively without or limited amounts of N fertilization, primarily due to economic constraints , and store C and N that can be remobilized after grazing, which are crucial for rapid regrowth after severe defoliation ; however, research demonstrating persistence and productivity of C4 grasses under a stressful defoliation regime is scarce. This information is valuable because a great proportion of C4 grasses are cultivated under marginal management practices.According to In general, new cultivars were selected and released over the last 50 years with the main objective to increase herbage accumulation and nutritive value, and this selection may have altered plant morphology, physiology, and preference by animals, which may negatively impact persistence. Vendramini et al. (2010) conducted a grazing trial to test the effects of different grazing frequencies on newly released cultivars of bahiagrass (“Tifton 9” and UF Riata) under a limited N fertilization regime. It was observed that Tifton 9 and UF Riata bahiagrass cultivars decreased root-rhizome mass and ground cover when grazed at 2 wk when compared to a 4 wk regrowth interval; however, Tifton 9 had greater root-rhizome mass than Argentine, Pensacola, and UF Riata when grazed at a 4 wk regrowth interval. It is recommended that new cultivars should be tested under a range of management practices, thus clear management guidelines are defined upon the release of the cultivar.Stocking rate is the most common animal-based measure of grazing intensity and it is suggested that it is more important than any other single grazing management decision . The mai4 grasses are known for their resilience under stressful defoliation management, few studies in the literature have reported the deleterious effects of grazing intensity and stocking rates on C4 grasses’ persistence. Although C4 grasses have not been explored in research projects. 4 grassland persistence, the choice of grazing method may not impact plant resilience, as long as the stubble height is maintained at the optimum levels for the specific C4 species. Stocking method is a defined procedure or technique to manipulate animals in space and time to achieve a specific objective . Stockin4 grasses have potential to increase CO2 mitigation due to the efficient photosynthetic pathway and superior biomass accumulation. In addition, a greater proportion of the biomass accumulation in C4 grasses occurs below ground, increasing C concentration in the soil  and control (plots with no N fertilization). Approximately 7% of the N2O-N emissions in grasslands are from N fertilizer sources, while 54% are from animal excreta ( excreta .4 grasses have usually greater cell wall and lignin concentration than C3 plants (4 grasses, such as concentrate supplementation, inclusion of legumes in the pasture, and use of additives, such as tannins and ionophores, among others (In spite of the potential greater C assimilation, C3 plants , which m3 plants . Howeverg others .4 grasses in regions previously occupied by C3 plants. This expansion is crucial to help to mitigate global warming due to C4 grasses’ efficient CO2 uptake and the ability to accumulate a greater proportion of the biomass below ground (4 grasses may dictate the adaptability of the species to specific edaphic-climatic-management conditions. Proper soil nutrient and grazing management promote forage production, nutritive value, and persistence, in addition to enhancing C4 grasses’ greenhouse gas mitigation potential.In summary, the rise in global temperatures favors the expansion of Cw ground . Differe