Aquimarina spongiae DMS (AsDMS) suggests that the FPP substrate is located within theinterdomain created by the DDxxE motif of N-domain and DxDTT motifof C-domain. Biochemical analysis revealed two aspartate residuesof the DDxxE motif that might contribute tothe capture of the pyrophosphate moiety of FPP inside the catalyticsite of AsDMS, which is essential for efficient cyclization and subsequentdephosphorylation reactions. The middle aspartate residue of the DxDTT motif is also critical for cyclization. Thus, AsDMSutilizes both motifs in the reactions. Remarkably, the unique proteinarchitecture of AsDMS, which is characterized by the fusion of a HAD-likedomain (N-domain) and a terpene synthase β domain (C-domain),significantly differentiates this new enzyme. Our findings of thefirst examples of bacterial DMSs suggest the biosynthesis of drimanesesquiterpenes in bacteria and shed light on the divergence of thestructures and functions of terpene synthases.Natural drimane-typesesquiterpenes, including drimenol, displaydiverse biological activities. These active compounds are distributedin plants and fungi; however, their accumulation in bacteria remainsunknown. Consequently, bacterial drimane-type sesquiterpene synthasesremain to be characterized. Here, we report five drimenol synthases(DMSs) of marine bacterial origin, all belonging to the haloacid dehalogenase(HAD)-like hydrolase superfamily with the conserved DDxxE motif typicalof class I terpene synthases and the DxDTT motif found in class IIditerpene synthases. They catalyze two continuous reactions: the cyclizationof farnesyl pyrophosphate (FPP) into drimenyl pyrophosphate and dephosphorylationof drimenyl pyrophosphate into drimenol. Protein structure modelingof the characterized  Class I enzymesinitiate reactions through ionization of a pyrophosphorylated substrate,7 whereas class II enzymes initiate reactions throughprotonation of carbon–carbon double bonds.8 Both mechanisms produce carbocation intermediates thatundergo a carbocation cascade to generate various terpenoids. Comparisonof terpene synthases reveals that the protein architecture is modularin nature and can contain up to three domains, termed α, β,and γ.11 Single-domain enzymes are currently described foronly the α-domain microbial class I terpene synthases, whileother class I enzymes exhibit αβ or αβγarchitectures. The class II terpene synthases feature βγor αβγ architectures. Recently, a single β-domainclass II terpene synthase has been noted for the merosterolic acidsynthase,12 underlining the diversity ofterpene synthase structures.Terpenoids, as the most diverseclass of natural products, are distributed in all living taxa, includingplants, fungi, bacteria, and protists. Terpenoids are well-known fortheir wide range of biological and pharmacological activities.15 terpenoids, fromfarnesyl pyrophosphate (FPP).13 The overallα- or αβ-domain architecture, with the active sitein the α-domain, serves as a template for catalysis by an STS.This ensures that the substrate FPP and subsequent intermediates adoptonly those conformations resulting in the formation of the correctproduct(s).10 A large number of STSs havebeen characterized in plants15 and fungi;16 however, much less is known about STSs of bacterialorigin, given the relatively small number of characterized bacterialsesquiterpenes.17 Indeed, drimanes, oneof the unique sesquiterpenes displaying antimicrobial, antifeedant,and insecticidal effects,18 are known fortheir predominant distribution in plants19 and fungi;20 however, the extent of accumulationof these active compounds in bacteria remains unknown. Consequently,no reports exist on bacterial drimane-type STSs, which are key biosyntheticenzymes of drimanes. Drimenol serves as the central biosynthetic precursorof various naturally occurring drimane sesquiterpenes,19 and it is a strong broad-spectrum antifungalagent.21 To date, two drimenol synthases(DMSs) of plant origin have been identified in valerian 22 and water pepper (Persicaria hydropiper).23 Therefore, it is important to discovernew bacterial drimane STSs, including DMSs to identify novel sesquiterpenesin bacteria.Sesquiterpene synthases (STSs)catalyze the biosynthesis of structurallydiverse sesquiterpenes, a family of CAquimarina spongiae DMS(AsDMS) enabled the characterization of the catalytic mechanism ofdrimenol biosynthesis and suggested a unique domain architecture ofthis new enzyme.Here, we report the functional characterizationof five DMSs ofmarine bacterial origin, all of which catalyze the biosynthesis ofdrimenol from FPP. The DMS enzymes contain two universally conservedaspartate-rich motifs of class I and II terpene synthases, which arerare for STSs. Using protein structure modeling and site-directedmutagenesis, AstC, a fungal drimane STS genefrom Aspergillus oryzae, belongs toa haloacid dehalogenase (HAD)-like hydrolase superfamily.20 Therefore, we searched a bacterial protein database24 for AstC homologues using a hidden Markov model(HMM).25 This analysis provided 1252 candidatesequences from bacteria; the candidates were then subjected to multiplesequence alignments. It was reported that AstC conserves a typicalDxDTT motif found in class II diterpene synthases. Accordingly, weanalyzed the 1252 candidate sequences and extracted only those containingthe DxDTT motif. Finally, four sequences were retrieved from bacterialclades as putative candidates. We then searched the National Centerfor Biotechnology Information (NCBI) database using the Basic LocalAlignment Search Tool (BLAST), with the four candidate sequences asqueries, and found two additional homologues from different bacteria.Six putative DMSs were obtained from Aquimarina aggregate (AaDMS), A. spongiae (AsDMS), Aquimarina sp. AU474 (A474DMS), Aquimarina sp. AU119 (A119DMS), Flavivirga eckloniae (FeDMS), and Rhodobacteraceae KLH11(RbDMS) (Thepreviously reported (RbDMS) 1.The si8-tag) in Escherichia coli. The three recombinant proteins  were visiblyexpressed in soluble fractions , and these soluble proteins were further isolated from the cellsby cobalt-immobilized metal affinity chromatography . Although the expression of the remaining three candidates was mainly observed in the pellet , we attempted to isolate them fromthe supernatant and successfully obtained purified proteins albeittheir low protein amounts .Toassess the catalytic function of the six DMS candidates, we expressedthe recombinant proteins fused with the N-terminal octa-histidine-tag to check whether thesesix candidates could dephosphorylate FPP or the reaction products.The products were analyzed by gas chromatography–mass spectrometry(GC–MS). As a result, the reactions of five proteins  with FPP yielded a single compoundcorresponding to the authentic (−)-drimenol (drim-7-en-11-ol),which is a sesquiterpene alcohol, in terms of the retention time (17.047min) and exact mass fragmentation pattern (parent mass m/z = 222) .To examine whether thesix candidate proteins had DMS activity,we performed z = 222) A,B. Thisive DMSs 2D and 3Cive DMSs 4, in theive DMSs 2B and 3Aive DMSs 2E,F. Theive DMSs 4. In addin vitro reactions ; however,geranyl geraniol, the precursor of diterpenes, was not observed withthe GGPP substrate . Similar observations were noted for AaDMS  due to the BAP activity.Next, to check whether the five DMSs have high specificities forFPP as a substrate, we performed the enzymatic reactions of theseproteins using geranyl pyrophosphate (GPP) or geranylgeranyl pyrophosphate(GGPP) as substrates. No reaction products were detected in the eactions 2D and 3CFigure S5). The half-maximal saturation concentration  for FPP, turnover number(kcat), and kcat/Km values were 9.59 ± 2.15 μM,0.086 ± 0.01 s–1, and 0.009 ± 0.001 s–1 μM–1, respectively. Thisresult indicates that AsDMS encodes a catalyticallyactive STS, and its kinetic properties agree with those reported forknown bacterial STSs.28 Notably, the catalytic efficiency of AsDMS is remarkablyhigher than that of plant-derived V. officinalis DMS, with both enzymes having similar Km values.22 This might result from thefact that plant STSs involved in secondary metabolism have low kcat/Km values inthe range of 0.001 s–1 μM–1 or even lower.29To thebest of our knowledge, this is the first report on drimaneSTSs of bacterial origin, which catalyze the cyclization of FPP into(−)-drimenol. We further characterized AsDMS that was highlyexpressed in the supernatant. The Michaelis–Menten kineticparameters were calculated by quantifying the amount of the drimenolproduct using GC–MS. A saturation curve was obtained from theMichaelis–Menten curve of the AsDMS protein . The crystal structures ofthe HAD-like phosphatase YihX from E. coli (PDB ID: 2B0C chain A)32 and ent-copalyldiphosphate synthase PtmT2 from Streptomyces platensis (PDB ID: 5BP8 chain A)33 were suitable template structuresfor homology modeling of the N- and C-domains, respectively. We nextassembled these domain models into the entire AsDMS model using domain-enhancedmodeling (DEMO)34 and predicted the spatialarrangements of the active site-defining residues within the AsDMSstructure.To obtain useful insightsinto the structural basis of AsDMS protein,we constructed a homology model of the AsDMS protein using the automatedSWISS-MODEL195–199 motifat the N-domain and DxDTT331–335 motif (a variationof the DxDD motif)36 at the C-domain were positioned inside the AsDMS model. These motifsare highly conserved among class I and II terpene synthase enzymes,respectively. It is interesting to note that a large cavity pocketexists between the N- and C-domains. We therefore speculate that thispocket is a substrate-binding site for FPP. To further investigatethe spatial position of the substrate-binding pocket in AsDMS, virtualdocking of FPP onto the AsDMS model was performed by AutoDock Vina.37 The docked structures were analyzed, and enzyme–ligandinteractions were observed. The results demonstrated that the FPPsubstrate fits within the interdomain shared by the DDxxE195–199 and DxDTT331–335 motifs  and theDxDTT331–335 motif (D333N). The recombinant AsDMSmutant proteins were expressed in E. coli and further purified  and thus enhances the acidity ofthe motif, exhibited almost equipotent activity to that of the WT,with and without the inclusion of BAP. This observation indicatesthat the positive charge of the basic residue K197 plays no role incatalysis by the DDxxE195–199 motif. Furthermore,the D195N/D196N and D195A/D196A variants, which were obtained by mutatingthe two aspartate residues to asparagine and alanine, respectively,entirely lost their DMS activity, although the D196N mutation lackingonly the second aspartate did exhibit some activity  was not detected despite the addition of BAP to the reactionmixture to compensate for the loss of dephosphorylation activity.This result indicates a complete loss of cyclization activity; therefore,we conclude that the N- and C-domains of AsDMS share a single activesite for sequential cyclization and dephosphorylation reactions  and a terpenesynthase domain (C-domain) was the new evolutionary target of theHAD superfamily and terpene synthase enzymes.AsDMS is characterized by an N-terminal domain thatinvolves a HAD-like domain (residues 30-214) comprising a DDxxEE. coli (accession: P0A8Y3)32 D sequence; its acidic aspartateresidues and those of motif I are important to coordinate the Mg2+ in the active site.45 From theseobservations, we propose that the N-domain of AsDMS involving theHAD-like domain shares a common evolutionary origin with HAD-likehydrolases and acts as a HAD phosphatase by catalyzing the dephosphorylationreactions. Interestingly, the HAD motif IV conserved in AsDMS is thefunctional DDxxE195–199 motif that is also commonlyfound in class I terpene synthases and responsible for positioningthe FPP substrate for further reactions.We traced thephylogenetic relationship of the N-terminal domain of the AsDMS enzymewith functionally characterized HAD-like hydrolases. Notably, theN-terminal AsDMS clustered with known bacterial HAD phosphatases andwas homologous to a phosphatase enzyme from 0A8Y3)32 7A. It ismotifs45 8A. TheN47 Given that the functional DxDTT331–335 motif located at the C-domain of AsDMS is alsoconserved among class II diterpene synthases and that class II enzymesconserve the DxDTT or DxDD motifs in a β-domain,49 we constructed the phylogeny of the C-domain of AsDMS with terpenesynthase β-domains. The results demonstrate that the C-domainAsDMS was homologous to the β-domains of bacterial class IIor plant terpene synthases  andC-domain (terpene synthase β domain) of AsDMS, each evolvedfrom different enzymes and uniquely fused 8C, substproteins8C.A. spongiae AsDMS was further characterized as abifunctional STS that utilizes two active DDxxE (N-domain) and DxDTT(C-domain) motifs for catalyzing sequential cyclization and dephosphorylationreactions to produce the final product, drimenol, from FPP. Notably,both HAD-like domain (N-domain) and terpene synthase β domain(C-domain) of AsDMS with different evolutionary origins might havebecome fused and formed an active site at the interface of two domains,giving rise to a unique protein architecture. Our discovery of bacterialDMSs paves the way for future studies on drimane sesquiterpenes inbacteria and highlights the diversity in the structures and functionsof terpene synthases, which play increasingly significant roles interpene biosynthesis.We identified five active DMSs of marinebacterial origin for the first time. Among the five DMSs, the 25 and the NCBI database. The six selected proteins were subjectedto multiple protein alignments to confirm the presence of the conservedDxDTT motif.CandidateDMSs were identified by screening all putative proteins against theHMM of AstC as a query, with HMMER web serverMethod S1.Details are provided in DMS genes were codon-optimizedfor expression in E. coli and synthesizedby GENEWIZ . Each sequence was amplified by the polymerase chain reaction(PCR) using PrimeSTAR GXL DNA polymerase with gene-specific primers1–12 (Table S1). The PCR productswere purified using a FastGene gel/PCR extraction kit , according to the manufacturer’s instructions,and further integrated into the linearized pET28b(+) expression vector(Nde I and Xho I) in-frame withthe N-terminal His8-tag by In-Fusion cloning . The sequence and correct orientation of the DMS genes were confirmed, followed by transformation into E. coli BL21 Star (DE3). The empty pET28b-His8 vector was used as a negative control for protein expression.The codingsequences of six candidate AsDMS was performedin the pET28b-His8AsDMS clone by inversePCR with gene-specific primers 13–24 (Table S1). Sequence determination of these mutagenized constructswas performed prior to expression.Site-directed mutagenesis of 8-tagged DMS proteins, including AsDMS, wereexpressed in E. coli BL21 Star (DE3)strains harboring pET28b-His8DMS and purifiedusing TALON metal affinity resin ,as described in Method S2. The concentrationsof the purified proteins were measured with a protein assay kit  using bovine serum albumin as a standard. The expressionand purification of AsDMS mutant proteins were performed as describedfor the recombinant AsDMS. These purified proteins were further usedfor in vitro enzymatic activity assays.All recombinantN-terminal His2, 1 mM dithiothreitol, and 100 μM FPP, GPP, or GGPP.Reactions in the absence of proteins were used as negative controls.The reaction mixture (400 μL) was incubated at 30 °C for1 h. Then, BAP from E. coli C75  was added to 200 μL of the reactionmixture and further incubated for 1 h at 37 °C. The aqueous layerwas then extracted twice with 200 μL of hexane/ethyl acetate. After centrifugation at 4400g for 5 min(4 °C), the upper organic layer was removed, and the combinedextracts were subjected to GC–MS analysis. The DMS proteinassays with farnesol as a substrate  were performedin the same manner as for the characterization of the DMS proteins,except for the absence of BAP treatment.For the initial characterizationof the DMS proteins (WT and mutants),the reaction was initiated by the addition of purified proteins  to a mixture of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2, 1 mM dithiothreitol,0–120 μM FPP, and 200 nM purified proteins at 30 °Cwithin 0–15 min (during which the enzyme activity was linear).The reactions were terminated by adding 20 μL of 2.0 M HCl.A 10 μL aliquot of (+)-nootkatone  was added. The products were extracted twiceusing 200 μL of hexane/ethyl acetate  and centrifugedat 4400g for 5 min (4 °C). The resulting organiclayer was combined and used for the GC–MS analysis.For the kinetic assayof the AsDMS (WT) protein, several reaction conditions were tested:a total volume of 200 μL of the reaction mixture containing50 mM Tris–HCl (pH 8.0), 2 mM MgCl–1 andmaintained for 10 min at 230 °C, followed by an increase at arate of 20 °C min–1 to 300 °C. Heliumwas used as the carrier gas at a flow rate of 1 mL min–1. The amount of the reaction product was measured as the peak areausing MSD ChemStation G1701EA E.02.02.1431 GC/MS software (Agilent).The results obtained from three independent experiments are expressedas means ± standard deviation (SD). The kinetic parameters werecalculated by fitting a Michaelis–Menten curve to the raw kineticdata using Prism 6.0h software . The raw data were calibrated in advance using a standardcurve of authentic (−)-drimenol.The reaction products were detectedwith a 7890 GC  coupledwith a 5975 mass selective detector and HP-5ms UI capillary column(30 m × 250 μm × 0.25 μm). A 1 μL samplewas injected in splitless mode at an inlet temperature of 260 °C.The initial oven temperature of 70 °C was increased after 1 minto 230 °C at a rate of 8 °C min30 pipelineusing the crystal structures of the HAD-like phosphatase YihX from E. coli (PDB ID: 2B0C chain A)32 and the ent-copalyl diphosphate synthase PtmT2from S. platensis (PDB ID: 5BP8 chain A)33 as template structures, respectively. Thesestructural models were assembled using DEMO software,34 and a full-length model of AsDMS was obtained. The AsDMSmodel was also predicted using AlphaFold2.31 The entire docking process was performed using AutoDock Vina 1.1.2software37 with the PyRx-0.8 interface.50 The 3D conformation of input ligand FPP wasobtained from PubChem (http://pubchem.ncbi.nlm.nih.gov/), and the geometry of theFPP structure was refined to minimize its free energy state usingAvogadro 1.2.0 software.51 The enzyme–ligandcomplexes with the highest docking scores acquired from the dockingprocess were obtained, and enzyme–ligand interactions werevisualized using PyMOL 1.8 . In addition, the potential conformers of FPP in the AsDMS protein,and the minimal distances between FPP and its contacting residues,were calculated using PyMOL.The three-dimensional (3D)structures of the N- and C-terminal domains of the AsDMS protein werepredicted by the SWISS-MODELhttps://www.genome.jp/tools-bin/clustalw), and the alignment was colored using the BoxShade 3.21 server . Evolutionary analyses of full-length amino acid sequences of theN- and C-terminal domains of AsDMS with other reported HAD-like hydrolasesand terpene synthase β domains, respectively, were performedin MEGA 7.0,52 based on a neighbor-joiningmethod using the p-distance algorithm with 1000 bootstrapreplicates.A multiplesequence alignmentwas generated using the GenomeNet ClustalW 1.83 server (


Background/Objectives: Yee Hong Play Intervention for Dementia (PID) is a community program strengthening East Asians >65 years with dementia in their daily functional activities. We analyzed how PID activities align with Hong Kong Montreal Cognitive Assessment. Methods: Utilizing observation sheets procured from documentation notes from the twice-weekly PID sessions, cognitive domains were identified. Mean time duration and activity frequencies were compared between high and low competency client groups. Results: Independent of competency group, activities predominantly targeted attention/concentration , and hand-eye coordination . Less focused domains were delayed recall (3.1–4.7%) and naming (1.3–1.5%). Conclusions: Yee Hong PID tested innovative cognitive domains not currently covered in HK-MoCA screening assessment, emphasizing attention/concentration-oriented activities and none assessing orientation and language domains. Additionally, presence of new domains such as hand-eye coordination and fine motor dexterity suggested that strict adherence with standardized screening tools  may not be ideal. Likely, facilitators have developed innovative measures to assess individual competency to strengthen resilience in our geriatric population. Cognitive assessments are typically conducted to evaluate and diagnose individuals who may be at risk or are currently affected by cognitive impairment . Mild coDespite confidence in using standard screening tools, the aforementioned studies put particular emphasis on comparison studies and test validation in conducting MoCA. New assessment tools are continuously suggested to accommodate and enhance detection of MCI, including Hong Kong Brief Cognitive Test (HKBC) to supplement HK-MoCA . BesidesMethods). Each group is then presented a variety of simulated activities that combine physical toys, modified game sets and miscellaneous materials  which PID-trained staff and volunteers creatively design. As per PID guidelines, the time limit for each activity is 8 minutes to maximize client engagement before moving to the next activity (p = .028) in MoCA scores specifically in the memory domain between geriatric clients in PID-treated group versus their control group (no PID treatment) (PID (Play Intervention for Dementia)  is a proactivity . Their mactivity . Additioeatment) . Furthereatment) . HoweverHK-MoCA will be our cognitive screening tool of focus given our predominantly Chinese and Oriental Asian clientele at Yee Hong. Over a 11-month period , our study objective analyzed how current PID activities align with standard screening tools such as HK-MoCA with respect to their cognitive domains, and further explore any recurring domains reflected in PID activities that were less thoroughly addressed in HK-MoCA. Additionally, we believe that specific cognitive domains may be more significant when assessing populations marked by differences in cultural and ethnic backgrounds . AdditioOur research question is as follows: Do PID activities at Yee Hong Centre for Geriatric Care (Mississauga) address the cognitive domains reflected in HK-MoCA? We are conducting a retrospective evaluation of program activities, comparing their assessed domains to HK-MoCA . Our goal is to identify if significant differences exist between the characteristics of Yee Hong PID activities conducted for the “High Competency” compared to the “Low Competency” client groups, whether it between frequency of domains, or duration per domain-specific activity.Yee Hong Centre for Geriatric Care (Mississauga) is a non-profit long-term care facility which provides a spectrum of services ranging from community-based recreational activities for clients age 50+ , with more focus toward intervention groups in the form of Adult Day Program, to housing services and long-term care.Within each PID session, there are two client groups subjectively differentiated by the PID Program Facilitators as: a) High Competency (HC) and b) Low Competency (LC) to create approximately similar clientele in terms of cognitive functioning. Competency was defined as a surrogate measure, based on extent of assistance , and overall mobility including use of wheelchairs.Separate assessments of data were conducted for each competency group. Furthermore, this observational study did not include direct identifiers or performance of individual clients because as mentioned previously, the aim is to strictly assess the cognitive domains addressed in PID activities and their respective time durations rather than assessing individual client performance. Specifically, client engagement was measured as amount of time spent per activity or number of clients attending this activity.This protocol was reviewed under the University of Toronto Health Sciences Research Ethics Board (REB) and approved on July 5th, 2019. The collection period lasted from January 4 to December 6, 2019. Four key components were analyzed in detail from these observation sheets:1) Assessed Cognitive Domain in each Activity2) Duration (minutes) of each Activity3) Outstanding Notes on Effectiveness of Activities Statistical analysis was conducted using software tools including Excel and RStudio . Session activities were aligned to a set of cognitive domains reflected in HK-MoCA. These domains include: Visuo-constructional Skills (VS), Naming (N) or the ability to refer to an object, person, place, concept or idea by its proper name, Memory (M), Attention & Concentration (AC), Language (L), Abstraction (A), Delayed Recall (DR), and Orientation (O), along with unique domains not reflected in HK-MoCA including Creativity (C), Fine Motor Dexterity (FM), and Hand Eye coordination (HE).t-tests were performed to measure differences between mean duration of each activity irrespective of domain between HC and LC groups. Follow-up two-sample t-test analyses were then conducted for each cognitive domain to measure differences in mean duration between HC and LC group activity. Secondary analysis included separate one-way ANOVAs on each competency group to compare mean time durations per domain, followed by two-way ANOVA to measure significant differences in mean time duration per domain across groups.Two-sample t-tests were performed to compare mean duration per domain in both groups to the null hypothesis represented by a recommended SSLD standard of 8 minutes per activity  to an expected distribution .Finally, separate χIn summary, we interpret our findings to align with the following objectives:a) Which cognitive domains were covered most and least extensively at the end of collection period?b) Any domains presented in PID that are not specifically reflected in HK-MoCA?c) Noticeable patterns that affect client engagement to staff/volunteer-led activities? ?d) Variances in instruction or performance time across specific domains (and do these durations change between competency groups)?e) Does mean duration of each domain-associated activity follow recommended guidelines listed in PID resource guides?A total of 85 sessions  documenting 1424 activities  and theit-tests, mean activity durations were compared between HC and LC groups. PID staff and volunteers did not follow the 8 minutes per activity standard and exceeded these limits for both HC and LC groups  (p = .0417). Please refer to t-tests between HC and LC groups (Through two-sample ctively) , signifiC groups  yielded p = .039). However, post-hoc tests (n < 5) in observation sheets.Separate one-way ANOVAs measuring mean duration across domains in HC and LC groups  yielded oc tests  revealedp < .0001), “Attention/Concentration” , “Creativity” , “Fine Motor Dexterity” , “Hand-eye Coordination” , “Memory” , and “Visuospatial”  were significantly longer than the recommended 8-minute limit. Under LC group, domains significantly longer than the recommended limit included “Abstraction” , “Attention/Concentration” , “Creativity” , “Fine Motor Dexterity” , “Hand-Eye Coordination” , and “Memory” .Single-sample t-tests were conducted comparing each mean duration from both LC and HC groups to 8 minutes (null) as observed in 2 Test of independence was conducted and showed significant association between competency group and their corresponding frequency domain count . The result was a significant difference from expected for both HC (p = .0013) and LC (p < .0001) groups.χp-values (p < .0001), Delayed Recall 3.1% , Hand Eye Coordination 19.12% , Naming 1.29% , and Visuospatial 6.46% . Post-hoc binomial tests for LC group revealed significant differences in: Attention/Concentration 16.42% , Delayed Recall 4.66% , Hand Eye Coordination 28.68% , Naming 1.47% , and Visuospatial 6.62% .Multiple post-hoc binomial test comparisons were subsequently conducted with Bonferroni’s correction, to adjust p-values  between p = .0417). When comparing mean duration per domain between HC and LC groups, there were no significant differences. When compared to the recommended 8 minutes per activity, 7/8 domains were significantly longer—abstraction ; attention or concentration ; creativity ; fine motor dexterity ; hand-eye coordination ; memory , and visuospatial (HC +6.89 minutes). When compared to their expected distribution , frequency of two domains were significantly greater: Attention/Concentration and Hand-Eye domains irrespective of competency group. However, within both groups, Delayed Recall, Naming, and Visuospatial (LC only) were all significantly lower than their expected counts.Over January to December 2019, two response variables were closely analyzed between HC and LC groups: a) mean duration and b) domain. HC groups spent significantly longer times carrying out each activity , we can attribute these differences to the nature and complexity of the activities. For example, abstraction-related tasks include matching shapes, colors, and card pattern recognition. Naturally, these tasks require a longer time for clients to complete. Furthermore, creativity  and hand-eye coordination  activities require less cognitive capacities, but provide an avenue for clients to express themselves through what they construct. This results in greater engagement when carrying out creative and hand-eye oriented activities.The same can be expressed in attention/concentration, hand-eye coordination, and memory domains for the LC group. However, it is more likely that the memory domain is due to LC client inability to effectively perform these tasks. Therefore, contrary to ending the activities early, corner facilitators may find themselves spending excess time to better guide these clients to recalling past memories to help them complete tasks. This adjustment in time can be paralleled to a study suggesting that MoCA threshold cut-offs be lowered to accommodate for clinics specifically addressing clients with memory disorders/impairment .Both HC and LC groups predominantly covered Attention/Concentration  and Hand-Eye  domain-related activities the most while Delayed Recall , Naming , and Visuospatial  domains were significantly lower. Due to constant adjustments made in PID to better suit client needs, we believe PID staff and volunteers are intentionally prioritizing “Attention” and “Hand Eye” domain-oriented activities because they elicit greater client engagement and social interaction between clients. Such games include Jenga, and throwing/reactionary games . However, there is a trade-off in other important domain-related activities such as “Delayed Recall” and “Naming” which are concurrently covered less. We also note that three additional domains not covered in MoCA  were extensively represented at an equal or higher than expected count (null = 1/9). We believe PID has made these domains known in response to the cultural differences in a program with predominantly East Asian clients , 2018. TAdditionally, our 11-month collection range (January to December 2019) may not exhibit the full spectrum of MoCA domains albeit our study reflected the most recent sessions within the community program. Furthermore, confounding variables such as client satisfaction, and intricate adjustments to activity function which may influence domain counts by PID staff and volunteers cannot be adequately measured.To comprehensively encompass the various domains within HK-MoCA, it is encouraged that PID observers to extend their evaluations beyond the activities and corresponding domains they assess. For instance, instead of pursuing language-oriented activities which may prove too difficult for low competency clients to engage in, language can be referred to the degree of social interaction and ability to understand basic instructions between PID client and peers and staff. Further, we highly encourage PID corner facilitators to incorporate more item-based  tasks and objectives to evaluate client in the “naming” domain. To better encompass the delayed recall domain which may not be practical, we suggest the use of icebreakers to encourage learning of client names or favorite hobbies. In this method, corner facilitators can ask clients to recall each other’s names and hobbies without compromising the facilitator’s activity sessions. We also hope that these new domains covered by PID can shed insight into the need to address cultural differences between clients during cognitive screening processes or programs to encourage proactive lifestyles in our geriatric community.


Key optimizations included pseudoproline incorporation, N-terminal hydrophilic tail addition and mild N-acetoxy succinimide acetylation. Their folding and activity are as for the recombinant protein. Our results will enable engineering of mirror-image monobody antagonists of the central oncoprotein Bcr-Abl.The  d- and l-versions of the Bcr-Abl SH2 domain (12.7 kDa) were synthesized successfully and show similar folding as compared to a recombinantly expressed version.The  Bcr-Abl is a deregulated highly active tyrosine kinase and can be potently inhibited by imatinib (Gleevec), the first molecular targeted cancer drug.1 Although imatinib and its successors have strongly improved survival of most CML patients, drug resistance mutations result in disease recurrence.2 Our previous work identified alternative approaches to target Bcr-Abl allosterically through disruption of an intramolecular protein–protein interaction of the Src homology 2 (SH2) domain and the kinase domain.3 This was achieved by engineered high-affinity monobody proteins, which bound the SH2 domain and inhibited Bcr-Abl activity, downstream signalling and leukemogenesis.4Bcr-Abl is the central driver oncogene of chronic myelogenous leukemia (CML) and subsets of B-cell acute lymphoblastic leukemia.5 SH2 domains are important drug targets, but the development of selective SH2-inhibitors, such as high-affinity peptides,6 peptidomimetics or small molecules, is notoriously difficult.7 In contrast, we developed various monobodies that bound different SH2 domains with nanomolar affinities and unprecedented selectivity.3,4,8–11 The lack of disulfide bonds in the fibronectin type III (FN3) monobody scaffold enables their activity in the reducing environment of the cytoplasm and resulted in inhibition of signalling of the target oncoprotein.11 The small size of monobodies (∼10 kDa) could show better tissue penetration and easier intracellular delivery into tumours. On the other hand, the clinical use of monobodies may be hampered by a low plasma half-life and induction of an immune response. A strategy to improve these properties relies on the generation of mirror-image monobodies consisting of d-amino acids. d-Proteins are non-immunogenic, metabolically more stable and have a longer half-life in vivo, because their amide bonds are not substrates of proteases.12,13 Engineering of d-monobodies requires synthesis of the target protein in d-configuration by solid phase peptide synthesis (SPPS) and native chemical ligation 32 and others33–35 enables protein synthesis beyond cysteine-containing ones.36 Since the Bcr-Abl SH2 domain lacks native cysteines, we envisioned a convergent two-segment synthesis where T197–A198 is the ligation junction  (1) and Abl1(198–241) (3), respectively  hampers its synthetic accessibility by routinely linear Fmoc based-SPPS.junction , and conectively . In the C-terminal thioester of the N-terminal segment 1, we used the 3-amino-4-(methylamino)benzoic acid (MeDbz) linker due to commercial availability, suppressed branching during amino acid couplings and mild activation procedure.37 SPPS of the C-terminal segment Abl1(198–241) (3) was straightforward (12% yield)  (1), and expected to worsen when biotin was coupled, which is required for target immobilisation during monobody selection. To overcome this issue, the addition of five lysine residues (K5) and a serine–glycine linker (SG) to the N-terminus resulted in great improvement of solubility (up to mM). Analysis of this N-terminal fragment after cleavage from resin revealed two truncations between I164 and N165 (3898.0 Da) and N165 and G166 (3783.9 Da). Gratifyingly, the incorporation of the corresponding glycine–serine (GS) pseudoproline -OH, PP) dipeptide38 at residues G166 and S167, and a gentle increase of the synthesis temperature to 50 °C completely suppressed the truncations , and hypothesized a possible acetylation on the secondary amine of the MeDbz linker because of the standard capping conditions ; C. elegans neuropeptide, where 76% of the MeDbz linker was acetylated by acetic anhydride.39 Besides, capping with acetic anhydride after every coupling step is generally not recommended with the MeDbz linker as it prevents the subsequent N-acyl-N′-methylacylurea (MeNbz) formation and NCL.40 However, removing this step could affect crude purity and aggravate peptide purification. Nevertheless, we successfully circumvented the formation of the acetylated side product without resorting to the removal of the capping step during synthesis by exchanging the acetic anhydride for the mild acetylation reagent with preference for primary amines, N-acetoxy succinimide  and DIPEA in anhydrous DMF the MeDbz linker of peptide 1 is converted to MeNbz (2). This acts as an efficient thioester precursor for the subsequent NCL reaction.37 Finally, peptide 2 was cleaved from the resin, HPLC purified and obtained with a yield of 10% -propane] dihydrochloride (VA-044).31 Nearly quantitative conversion was observed after 16 h at 37 °C. Thus, the purified full length Bcr-Abl SH2 domain (5) was obtained with a yield of 72% -OH, Fig. S14–S17, ESId-Bcr-Abl SH2 counterpart (d-5)  of ∼60 °C for the recombinant and a mildly higher Tm for the synthetic l- and d-Bcr-Abl SH2 domains were observed  showed that the synthetic l-Bcr-Abl SH2 domain bound AS25 with the same affinity as the recombinantly expressed protein  ligand peptides. A pY peptide that was previously shown to bind the Src SH2 domain10 bound the synthetic l-Bcr-Abl SH2 domain with the same affinity as the recombinantly expressed domain (d-Bcr-Abl SH2 domain, the corresponding d-pY peptide bound the d-protein with the same affinity as the l-pY peptide to the l-Bcr-Abl SH2 domain (d-pY peptide did not bind the recombinantly expressed l-Bcr-Abl SH2 domain (Fig. S20B, ESIWe next determined whether the synthetic  protein . Given td domain . To dete2 domain . As expeN- and C-terminal fragments of the Bcr-Abl SH2 domain could be efficiently synthesized after GS-pseudoproline incorporation and suppression of MeDbz linker acetylation by exchanging acetic anhydride in the capping solution with N-acetoxy succinimide. The efficiently ligated and subsequently desulfurized l- and d-Bcr-Abl SH2 domain polypeptides were refolded in their native conformation and have binding properties similar to the recombinantly expressed domain.In summary, the d-Bcr-Abl SH2 domain protein for monobody development by phage- and yeast-display selection. Mirror-image monobodies will be an important step towards therapeutic application due to their higher in vivo stability.We will use the The authors declare no competing interests.CB-003-D2CB00108J-s001


Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction.−/− and p35−/− neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1−/− neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination.Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.The online version contains supplementary material available at 10.1186/s12915-022-01312-4. Cyclin-dependent kinase 5 (Cdk5) mainly functions in post-mitotic neurons due to the neuronal expression of its dominant regulatory partner, p35 –3. Cdk5 Previously identified and characterized as a scaffold protein, JNK interacting protein 1 (JIP1) plays an essential role in axonal development and neurite extension. JIP1 specifically localizes to the axonal growth cones and its deficiency impairs axonal elongation during neuronal differentiation . MoreoveA particularly important interacting partner of JIP1 in this regard is Notch1. Notch1 controls axonal outgrowth and guidance in postmitotic neurons –14 and uSince our interactome study revealed JIP1 as a potential binding partner of p35, we investigated whether a functional interaction exists between Cdk5 and JIP1. We hypothesized that Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition. Here, we demonstrate that JIP1 is a novel substrate of Cdk5 and Cdk5-mediated phosphorylation of JIP1 at Threonine 205 (T205) promotes axonal outgrowth. Consistent with this observation, exogenous expression of a phosphomimic JIP1 T205D rescues the defects on axonal growth caused by Cdk5/p35 and JIP1 deficiency. A noticeable increase in Notch1-IC levels in JIP1-deficient neurons and the rescue of axonal outgrowth phenotype in these neurons by Notch1 inhibitor Numb point to a role of this signaling in JIP1/Cdk5-mediated axonal development. Importantly, JIP1 T205 phosphorylation by Cdk5 is mechanistically related to the upregulation of Cdk5-mediated Itch phosphorylation at Threonine 222 (T222) which enhances the ubiquitination and proteasomal degradation of Notch1-IC. Our results present a compelling delineation for the involvement of a novel Cdk5-JIP1-Notch1 axis in the regulation of axonal outgrowth.Data from our previous mass spectrometry-based interactome screen , 24 iden32P-ATP with glutathione S-transferase (GST) or GST fused with JIP1 (GST-JIP1) with activated Cdk5, in the presence or absence of Cdk5 inhibitor, Roscovitine (Rosco). Phosphorylation of JIP1 was observed when JIP1 was incubated with activated Cdk5, which was notably decreased in the presence of Roscovitine PX (K/H/R) [The interaction of p35 and JIP1 indicates that JIP1 is a potential substrate of Cdk5. Since Cdk5 phosphorylates Proline (Pro or P)-directed Serine (Ser or S) or Threonine (Thr or S) in its substrates, we examined whether Cdk5 can phosphorylate JIP1 at these sites. We performed a radioactive in vitro kinase assay by co-incubating To test whether T205 or S235 phosphorylated JIP1 regulates axonal outgrowth, CD-1 cortical neurons were infected with adenoviruses expressing GFP control (GFP), WT-JIP1, T205A, T205D, S235A, or S235D at the time of plating (days in vitro (DIV) 0). At DIV3, axonal length of GFP-positive neurons was measured. Axon lengths of neurons expressing T205A, but not S235A, were significantly shorter than those expressing control GFP. In contrast, expression of T205D significantly increased axon length   at the time of plating. Neuronal cells were collected at the time points described in each figure legend.Primary cortical neuronal culture was carried out as described previously . Briefly2O and mixed with trace amounts of Trypan Blue dye. The plasmid was injected into the lateral ventricle of the brain using an Eppendorf Femtojet Microinjector and electroporated into the dorsolateral cortex using a BTX ECM 830 Square Wave Electroporator. Animals were sacrificed as described in figure legend. For analysis, electroporated sections at equivalent rostro-caudal levels of the brain, relative to the corpus callosum and anterior commissure, were selected.In utero electroporation was performed as previously described . Briefly125 (PO3) PPHEH, using standard protocols by PhosphoSolutions .Mouse anti-JIP1 , anti AIP4/Itch , Rabbit anti-p35 , Mouse anti-NUMB Antibody , Anti-c-Jun antibody , and anti-Notch1  were purchased from Santa Cruz Biotechnology . Mouse anti-GFP , Anti-ITCH/AIP4 antibody (Cat. #ab220637), Rb pAb to Myc tag , and Rb pAb to ubiquitin  were purchased from abcam . Rabbit anti-Notch1 , rabbit anti-Notch2 , rabbit anti-Notch3 , rabbit anti-Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) , monoclonal mouse anti-Phospho-Threonine-Proline (P-Thr-Pro-101) , SAPK/JNK antibody , Phospho-SAPK/JNK (Thr183/Tyr185) antibody , Phospho-c-Jun (Cat. #3270s), and p35/25 (C64B10) rabbit mAB  were purchased from Cell Signaling Technology . Mouse monoclonal anti-β-actin  was purchased from Sigma-Aldrich . Itch polyclonal antibody  was purchased from Invitrogen . Rb p-Itch (Thr222) antibody  was purchased from EMD Millipore . Special custom anti-phosphoJIP1 at Thr205 and anti-phosphoJIP1 at Ser235 were generated and purified from rabbit immunized with carrier protein-conjugated phosphopeptides, LKTGEQThttp://n2t.net/addgene:26891; RRID: Addgene_26891) [Human JIP1 was amplified by RT-PCR using RNA isolated from SH-SY5Y as template. The amplified JIP1 was cloned in pAdtrack-CMV vector. Mouse Itch construct (Cat. # R210764) was purchased from Origene  and Itch was subcloned into pCI-neo with 3x-myc. FLAG-Notch1-IC, a gift from Nicholas Gaiano  . JIP1 anThe binding assay was performed as previously described . BrieflyCdk5 kinase assay was performed as previously described . BrieflyImmunoblotting was performed as previously described . BrieflyImmunofluorescent (IF) staining for brain tissue was performed as previously described . EmbryonAxonal length from neuronal cell culture was measured at 3 days after plating and the GFP positive longest neurite of primary neurons was defined as the major axon as previously described . The meaIP was carried out as previously described . Briefly6 HEK293T cells  in a 60-mm dish were transfected with 3 μg of FLAG-Notch1-IC in the presence of 6 μg of empty vector or Itch-Myc or Itch mutants utilizing 9 μl of Lipofectamine 2000. Twenty-four hours after transfection, the transfected cells were treated with 10 μM MG132 for 4 h. The total proteins were extracted in 100 μl of SDS sample buffer , 1% Glycerol). After 5 min of boiling and 20 min of centrifugation  at 4 °C, the supernatants were diluted 10 times with water for IP using 2 μg of Notch1 antibody and 30 μl of anti-mouse IgG beads (eBiosciences) for 2 h at 4 °C. The beads were washed four times using a wash buffer containing 20 mM Tris (pH 7.4), 300 mM NaCl, 0.1% SDS, and 1 mM EDTA. Proteins were eluted in 2× SDS sample buffer for separation by 4–15% SDS-PAGE gel (Bio-Rad) and Western blot analyses utilizing an anti-ubiquitin antibody.Ubiquitination assay was performed as previously described . BrieflyFor the ubiquitination assay of endogenous Notch1 using JIP1 WT and KO cortical neurons, cortical neurons at DIV 3 were harvested in lysis buffer and IPed with 2 μg of Notch 1 antibody. Western blot was performed with an anti-ubiquitin antibody. The assay was performed like that described above.t-test was used to compare two groups.All statistical analyses were employed by using GraphPad Prism software version 8.0 (RRID: SCR_002798). In this study, no blinding or randomization was performed. Data are expressed as the mean ± SEM. One-way or two-way analysis of variance (ANOVA) was used to compare multiple groups. Tukey test was used for post hoc comparisons after ANOVA. Unpaired two-tailed Student’s Additional file 1: Table S1. Primers for JIP1 and Itch constructs.Additional file 2: Fig. S1. Specificity of anti- phospho-JIP1 antibody. Fig. S2. Notch2-IC or Notch3-IC level was unaffected by JIP1 deficiency. Fig. S3. Numb, Itch and phospho-JNK level were unchanged by JIP1 deficiency. Fig. S4. Phopho-Itch at Thr222 enhances interaction with Notch1-IC.Additional file 3. Western blots.Additional file 4. Individual data values.


The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a ‘super depolymerizer’ and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells. Kinesins are found in all eukaryotes, with 45 kinesin genes present in the human genome . The chaPhylogenetic analysis of the kinesin superfamily has identified 17 individual families, many of which can be further divided into subfamilies ,10. The α/β-tubulin heterodimer, resulting in destabilization of the microtubule. Structures of the motor domain of either KIF2A or MCAK in complex with the α/β-tubulin heterodimer show that they promote curvature of tubulin larger than that observed for similar complexes of tubulin with a kinesin-1 motor domain [The kinesin-13 family are a set of specialist microtubule depolymerases, with a centrally located motor domain flanked by divergent N- and C-terminal regions . Kinesinr domain –26. The r domain . The abir domain .By creation of reference motor domain sequences, we sought to capture the activity associated with the kinesin-13 family as a whole. The conceptual differences between the reference motors are non-trivial: the ancestral motor must have been functional but was not necessarily tuned to the specialized roles seen in extant cells. By contrast, the consensus motor could be either an optimal sequence for a family or a non-functional average. We expressed both the consensus and ancestral kinesin-13 reference motor domains and characterized their behaviour with respect to microtubules. Here we show that, while the consensus sequence acts as a microtubule depolymerase, its activity is reduced relative to MCAK. By contrast, the ancestral kinesin-13 motor domain is the fastest microtubule depolymerase studied to date, with a depolymerization rate more than an order of magnitude faster than MCAK.. 2. 2.1a). This latter ‘ancestral’ sequence was a maximum-likelihood estimate of the sequence existing at the root of the kinesin-13A and -13B subfamilies based on our previous phylogeny of kinesins [To comprehensively sample the sequence space occupied by the motor domain of a kinesin family, we generated reference sequences for the motor domain of the microtubule depolymerizing kinesin-13 family from an alignment of the motor domains present in a diverse set of eukaryotes . This mofigure 1kinesins  and the b). The MCAK neck is included as it is shown to be required for maximal depolymerization activity of the MCAK motor domain [c).We expressed and purified protein constructs containing these motor domain sequences with the addition of the neck region of the human kinesin-13, MCAK and EGFP for visualization b. The MCr domain ,29. Neit−1, which is approximately 33% of the rate observed for full-length human MCAK and approximately 50% of EGFP-labelled full-length MCAK . These data make the inferred ancestral kinesin-13 motor domain the fastest microtubule depolymerase observed to date.We measured the depolymerization activity of these kinesin-13 reference sequences, using GMPCPP-stabilized fluorescently labelled microtubules . Both thgth MCAK . The ancled MCAK a–c. Thesa). The depolymerization rate with ADP as the available nucleotide was 0.04 ± 0.01 µm min−1  and so is predicted to exist as a monomer. To determine if the increased depolymerization rate is due to the monomeric nature of the Anc13 motor domain rather than sequence changes within the motor domain, we created the MCAK-M construct in which the ancestral motor domain is replaced with the motor domain of human MCAK in the Anc13 construct (figure 1b). The microtubule depolymerization rate for this monomeric version of the MCAK motor domain is 2.11 ± 0.87 µm min−1 (figure 2a and p = 0.96) and approximately 11-fold lower than Anc13. These data indicate that, rather than differences in the oligomerization state between Anc13 and full-length MCAK, the increased rate of microtubule depolymerization for Anc13 is due to sequence changes in the motor domain.Full-length MCAK is a homodimer with dimerization occurring through the N- and C-terminal domains which flank the centrally located kinesin-13 motor domain . The Ancfigure 1. 2.2a and d) [−1 . Anc13, however, depolymerized double-stabilized microtubules at 1.01 ± 0.59 µm min−1 , 50-fold faster than the basal depolymerization rate and approximately 48% of the depolymerization rate of single-stabilized microtubules by full-length MCAK. These data show that the Anc13 motor is a more powerful depolymerase than MCAK, causing significant depolymerzation of microtubules stabilized by conditions which MCAK cannot overcome.Full-length MCAK can depolymerize microtubules stabilized either by incorporation of the slowly hydrolysable GTP analogue GMPCPP or by binding of the drug Taxol, but does not depolymerize microtubules stabilized by both GMPCPP and Taxol a and d [a and d) . Double-d d) [−1 , not sigd d) [−1 a,d,f. Anµm min−1 a,e, 50-f. 2.3a were collected at 8 nM MCAK, whereas the data shown in figure 3b were collected at 40 pM Anc13. We calculated a rate of attachment to the microtubule of 62.4 ± 21.5 nM−1 s−1 µm−1 for Anc13, which is approximately 100-fold higher than the microtubule on-rate previously determined for MCAK . Although lattice-interaction events for Anc13 are longer in duration, as for MCAK, they are diffusive interactions. The off rates for the microtubule lattice and microtubule end, calculated for Anc13 are similar, koff (lattice) = 1.36 ± 0.06 s−1 and koff (end) = 1.23 ± 0.10 s−1. The ratio of koff (lattice/end) for Anc13 is 1.11 ± 0.14, not significantly different from 1, indicating that, unlike MCAK, Anc13 does not discriminate between the microtubule end and the lattice.MCAK displays mostly short (less than 1 s) diffusive interactions with the microtubule lattice. Only at microtubule ends are longer lived interactions observed. Typically, approximately 30% of MCAK end-binding events last for longer than 2 s, whereas no lattice-interaction events longer than 2 s are observed . In agre. 2.4−1, which is approximately 200-fold higher than for full-length MCAK .To understand how the turnover of ATP is coupled to microtubule depolymerization by Anc13, we measured ATP turnover in solution, in the presence of unpolymerized tubulin and in the presence of GMPCPP-stabilized microtubules . The solgth MCAK . This hi−1, an increase of approximately eightfold over the basal ATPase. However, this fold increase in ATP turnover is small compared with MCAK for which unpolymerized tubulin causes an approximately 100-fold increase over the basal rate of ATP turnover [ATP turnover by Anc13 was accelerated by unpolymerized tubulin. The ATPase rate for Anc13 in.the presence of 10 µM tubulin was 3.76 ± 0.81 sturnover .−1, only an approximately 20-fold increase over the basal ATPase.The fold acceleration in ATP turnover caused by the presence of microtubules is also small for Anc13 compared with MCAK . While tThese data show that the ancestral kinesin-13 construct has a high rate of ATP turnover even in the absence of tubulin, whereas the MCAK motor domain placed in the same context, as part of the MCAK-M construct, has a very low basal ATPase similar to full-length MCAK. This indicates that the high basal ATPase of Anc13 is an intrinsic property of this motor domain, rather than a result of the absence of the N- and C-terminal regions that flank the motor domain in full-length MCAK.. 2.5a). This construct depolymerized GMPCPP-stabilized microtubules at 19.50 ± 4.87 µm min−1, approximately ninefold faster than the depolymerization rate for MCAK and not significantly different (p = 0.1) from the depolymerization rate observed for the Anc13 construct . These data indicate that the ancestral-13 motor retains ultra-rapid depolymerization activity when placed in the context of full-length MCAK, the addition of surrounding N- and C-terminal regions does not significantly alter its microtubule depolymerization activity.To understand the behaviour of the ancestral-13 motor domain within the context of a full-length extant kinesin-13, we created a construct in which the motor domain of full-length wild-type MCAK was replaced with the ancestral-13 motor domain, MCAK-Anc13 a. This construct b,c. Thesd,e). This activity is not frequently observed when microtubules are subjected to depolymerization by MCAK, which typically causes depolymerization only from microtubule ends. Quantification of the frequency of internal breaking for microtubules longer than 5 μm showed that in the presence of MCAK approximately 5% of microtubules (n = 58) broke at internal sites, while in the presence of MCAK-Anc13 approximately 70% of microtubules (n = 47) broke and began to depolymerize from these breaks at a similar rate as from the original microtubule ends. These data indicate that the ancestral motor is an extremely potent depolymerase both as a synthetic monomeric construct and as a dimer in the context of a full-length kinesin-13.In contrast with both MCAK and Anc13, the MCAK-Anc13 construct was observed to promote breaking of GMPCPP-stabilized microtubules leading to depolymerization from internal sites d,e. This. 3. 3.1The design and study of reference sequences for the kinesin-13 motor domain provide insight into the evolution of the kinesin-13 family and generalized information on the activity of the family as a whole. Both the consensus and ancestral motor domains have microtubule depolymerizing activity and depolymerize microtubules from both ends. The consensus sequence (Con13), which contains the most common residue at each position across the family, depolymerizes microtubules at approximately 1/3 the rate of our comparator MCAK. This agrees with the assumption that all members of the kinesin-13 family are microtubule depolymerases.In contrast with the consensus protein, the microtubule depolymerization rate for the ancestral kinesin-13 motor domain (Anc13) is an order of magnitude faster than the most highly studied member of the kinesin-13 family, MCAK. To our knowledge, this synthetic protein is the fastest microtubule depolymerase observed to date. In fact, Anc13 has such potent activity that it can depolymerize microtubules under stabilizing conditions in which MCAK has no effect. These observations suggest that the founding members of the kinesin-13 family had much more potent depolymerase activity than kinesin-13s currently existing in nature (or at least those studied to date). Of course, this activity may have been tempered in ancestral cells by tight regulation of expression or localization, or by inhibition by regions adjacent to the motor domain that can no longer be reconstructed by phylogenetic analysis. However, potent depolymerase activity is retained when the ancestral-13 motor domain is placed in the context of full-length MCAK surrounded by the non-motor N- and C-terminal regions. It appears that kinesin-13s in extant lineages have not evolved to become ‘better’ depolymerizers but have instead evolved away from ultra-rapid ‘super’ depolymerase activity towards milder action.. 3.2Single-molecule TIRF data show that, while Anc13 has higher overall affinity for microtubules, it does not distinguish between the microtubule end and the microtubule lattice. Rather, Anc13 displays long binding events at all positions on the microtubule. This contrasts with MCAK for which long binding events are observed only at or near microtubule ends , focusin. 3.3The stoichiometry of ATP consumed per tubulin removed by MCAK has previously been measured to range from 1 ATP to greater than 20 ATPs per tubulin . During The observation that the microtubule-stimulated ATPase for Anc13 was only approximately twofold higher than MCAK while the microtubule depolymerization rate was greater than 10-fold higher was unexpected, as this indicates that Anc13 removes more than a single tubulin for each cycle of ATP turnover. The stoichiometry of ATP consumed per tubulin removed for Anc13 is approximately 0.2 ATP per tubulin, indicating that for each ATP consumed Anc13 removes multiple tubulin dimers. From our data, we calculate that Anc13 removes on average 5.8 ± 1.4 tubulins per ATP, when acting on GMPCPP-stabilized microtubules.b) or, perhaps more likely, that Anc13 binds anywhere up to six dimers away from the microtubule end and causes destabilization of the bound tubulin leading to detachment of the remaining subunits from the same protofilament plus destabilization of tubulin subunits from neighbouring protofilaments, causing on average six dimers to detach from the microtubule (figure 5c).To explain these data, Anc13 must be able to bind at locations prior to the terminal tubulin dimer and destabilize tubulin subunits relatively distant to the microtubule end . Our datfigure 5Tubulin dimers farther from the microtubule end are probably more stably embedded within the microtubule than those close to the end. Therefore, to destabilize tubulin subunits at sites prior to the terminal dimer of a microtubule protofilament would require higher affinity binding to deform and destabilize tubulin dimers located at more stable positions within the microtubule. Anc13 not only binds microtubules with higher affinity than MCAK, but also can depolymerize microtubules under stabilizing conditions that cannot be overcome by MCAK. Further, when the ancestral-13 motor domain acts as a dimer in the context of full-length MCAK, it causes breaking of microtubules at internal positions. These data indicate that the ancestral-13 motor domain can promote deformation and destabilization of tubulin subunits distant to the microtubule end resulting in the removal of multiple dimers per ATP turnover cycle and internal breaking of microtubules, resulting in the most rapid microtubule depolymerase activity observed to date.. 3.4Our data suggest that the founding members of the kinesin-13 family were highly potent, ‘super’ depolymerases. The comparison of the activity of a phylogenetically inferred ancestral kinesin-13 motor domain with extant kinesin-13s implies that as the family developed its members evolved away from rampant depolymerization towards more controlled, but less rapid, activity. The comparison of the biochemical parameters for Anc13 with those previously determined for MCAK shows that while Anc13 can depolymerize microtubules an order of magnitude faster, this comes at the cost of efficiency in terms of ATP turnover and specificity in localization.It is not obvious which sequence changes between Anc13 and MCAK are responsible for the increased potency of the ancestral-13 motor domain. Sequence changes are scattered throughout the motor domain  and no sequence alterations are found in any of the conserved kinesin-specific nucleotide-binding motifs or in the kinesin-13-family-specific microtubule-binding motifs.In comparison to the ancestral sequence, modern-day MCAK has gained several characteristics that are presumably beneficial for the physiological roles of extant members of the kinesin-13 family. Mitotic spindle and flagellar length control are proposed to be the principal roles of kinesin-13s conserved throughout the evolution of eukaryotes . Highly . 4. 4.1Reference sequences for the kinesin-13 family were based on the previous analysis of 1624 kinesins proteins encoded in the genomes of 45 diverse extant eukaryotes . The aliConsensus motor domain sequence was inferred by a simple most common residue method, with ties resolved based on most likely residue by BLOSUM62 score. Ancestral sequences were inferred by maximum likelihood from the kinesin-13A/B subtree and alignment of extant motor domain sequences using the WAG substitution matrix as implemented by fastML v. 3.1 (–seqType aa –SubMatrix WAG –indelReconstruction ML) . A singl. 4.2Spodoptera frugiperda (Sf9) codon usage table . These coding sequences were incorporated into derivatives of pFastBac1 encoding the neck region from Hs MCAK on the N-terminal side and eGFP and TEV-cleavable streptavidin-binding peptide- and His-tags on the C-terminal side . The formation of the expected chimeric sequence was confirmed by Sanger sequencing covering the full CDS.Reference protein sequences were reverse translated using a 2, 0.1% Tween 20, 10 mM imidazole, 1 mM DTT and protease inhibitors. The proteins were purified by Ni-affinity chromatography  followed by StrepTactin affinity chromatography .The proteins were expressed in Sf9 cells . Cells were lysed by incubation at 4°C for 1 h in 50 mM Tris pH7.5, 300 mM NaCl, 10% glycerol, 5 mM MgCl. 4.3Porcine brain tubulin was purified from homogenized brain tissue using the high ionic strength method . Microtu. 4.4−1 BSA, 1% 2-mercaptoethanol, 40 mM D-glucose, 40 mg ml−1 glucose oxidase and 16 mg ml−1 catalase. Images were recorded using a Zeiss PS1 Super Resolution microscope. Microtubules were imaged using laser excitation (561 nm) and a 579–620 nm emission filter. Movies of eGFP-labelled single molecules were collected using TIRF excitation at 488 nm and a 495–550 emission filter, 200 images were collected per movie with a frame rate of 8.7 Hz.Rhodamine-labelled, GMPCPP-stabilized microtubules were adhered to the surface of flow chambers prepared as described previously . MCAK-eGFor analysis, each frame of the GFP dataset was colour-combined with the corresponding image of rhodamine-labelled microtubules in FIJI to enable identification of on-microtubule events. Kymographs for individual microtubules were used to measure the duration of individual GFP localization events at the microtubule end and on the lattice, also in FIJI .. 4.5ATPase rates in solution were measured using 3 µM kinesin by monitoring the production of ADP using HPLC as described previously . For ass


The domestic yak, a type of cattle native to the Himalayan region and Siberia, is known for its ability to withstand harsh climatic conditions at high altitudes and is primarily domesticated for its milk, wool, and meat. Most of the research on yaks has been conducted in China, where approximately 94% of the total estimated yak population can be found. In this study, the authors conducted an ethology study on the social behaviour of yaks in the Annapurna Valley of Nepal, where they are raised for their milk, wool, and meat and are also used for transportation. The authors used ethnographic inquiries to gather data on the social behaviour of yaks and the coping strategies used by herders to manage them. They also equipped cattle with one Actigraph wgt3x-BT to measure activity using an accelerometer and spatial associations using a proximity recorder. They found that yaks in both herds exhibited cohesive and synchronized behaviour, with similar activity patterns during the day and a peak of activity at night. They also observed that yaks in the first herd were more reactive to the presence of humans, while those in the second herd were more reactive to the presence of other yaks. The authors suggest that these differences in behaviour may be due to the different herding management practices used in each herd.Herdsmen use different techniques, as per varying geographies and cultures, to keep the cohesion within herds and avoid animals getting lost or predated. However, there is no study on the social behaviour of yaks and herdsmen management practices. Therefore, this ethology study was initiated by ethnographic inquiries. In Manang, the success of the shepherd is dictated by his personal attribute of ‘Khula man’ or open-heartedness. This attribute refers to good intentions and emotions such as empathy, which allow the shepherd to focus more on others than on himself. This cultural way of assessing the skills required to become a successful and knowledgeable shepherd guided us to study the effect of cultural values on the herd’s social behaviour. We collected data from two herds living at the same settlement  by equipping them with loggers. One of the herdsmen used the tether rope while the other one did not. Moreover, the Thaku herd had a more proactive shepherd than the Phurba one. In each herd, 17 animals were equipped with one Actigraph wgt3x-BT to measure activity using an accelerometer and spatial associations using a proximity recorder. One of the herds was equipped with GPS (N = 11) as well. Using GPS locations and activity, we showed that the two herds were cohesive and synchronised their activities but the Thaku herd (tether rope herd) was more cohesive than the Phurba herd based on the Actigraph signals. The shepherds also have personal knowledge of the social relationships of individual animals in their herds and use these relationships to keep the group cohesive and to manage cattle well. Bos grunniens), a descendent of wild yak (Bos mutus), is domesticated in the Himalayan region and in Siberia for its milk, wool, and meat but mainly for its tolerance to harsh climatic conditions at high altitudes [The domestic yak , and does not take punitive action, the herd stays around and comes back to the settlement. These shepherding skills impact herd behaviour and it is important to study the effect of human actions shaping the social behaviour in yaks.This study was conducted over a period of 10 months, beginning in 2017 and ending in 2018. For 10 months, we observed that by strategising facilitating habits or daily rites, the shepherds nurtured the cauri  to form a good herd. Shepherds emphasised on the quality of the relationship they had with every yak in the herd as it was crucial for daily milking. The quality of the relationship was tested over milking as milking was interpreted as the cooperation of the cauri that ‘gives her milk’ to the shepherd. To achieve this, the shepherd tied all the young ones to the tether rope in particular spots, irrespective of the season. The calves of the same age were grouped together to ‘make friends’ . Until three years of age, the calves were tied to the tether rope as they nursed on their mums, and the older ones took their places by themselves. The placement of each calf was chosen to prevent the juveniles and the mother yaks from fighting with each other. This helped the shepherd to find his herd in the morning, and to have a harmonious relationship at the milking station by reducing stress, improving the milk quality, and building cohesiveness amongst his herd. If not conducted this way, the calves would destroy the tether rope during the night and it would become impossible to get the herd back from the pastures, as they would not stay together or they would refuse to come back to the settlement and not cooperate. The daily movements of the herds in the pastures are implicitly related to the shepherd’s work and personality. If the shepherd is considerate, takes care of his cattle by giving The shepherds also have personal knowledge of the social relationships within their herds. They do not automatically assimilate a mother yak’s personality to her calf. The calves develop their own personalities with time. Friendships between the calves were frequent and beyond the shepherd’s control, e.g., two calves were too slow and always stayed together at the end of the herd, next to the shepherd. As frequent loneliness is a criterion of personal complexity or non-subjection and a sign of intelligence, yaks may need the cohesiveness that can be improved by the shepherds.In the winter of 2022, the data were collected from two herds living at the same settlement  by equipping them with loggers. One herdsman used the tether rope in his herd (named Thaku) whilst in the other herd (named Phurba), a rope was not used. Moreover, the Thaku herd’s shepherd was more proactive than the Phurba one. In each herd, 17 yaks were tagged with an Actigraph wgt3x-BT to measure activity using an accelerometer and spatial associations using proximity recorders . The proThe shepherds were excited and participated actively in fitting the loggers. They were curious at the possibility of observing their herd onscreen and seeing if the cauri were staying together or dispersing on the pasture. The equipped cattle were chosen by the shepherds, who categorised the animals in groups such as mothers and calves, good or bad ones, or two inseparable friends. The calves could not be equipped with the GPS as the loggers were heavy and some cauris could not be equipped because they were uncooperative. As a result, only the cauri that were closest to the shepherd were equipped.Two herds were shepherded by two different herdsmen at Yak kharka, Manang, Annapurna Valley, Nepal . During the study, at least four snow leopards were observed in the valley (from 10 to 24 March 2022). Moreover, a month before and after the study, four yaks were killed by the predator. A dog was used to keep the snow leopard away from the village. At night, the dog stayed in the village and during the day it could roam between the village and the pastures. In each herd, 17 yaks were equipped with Actigraphs ,13 but GAll loggers of yaks were synchronised and every herd started at the same time.Activity: Activity was measured using an accelerometer with the Actigraph wGT3X-BT [2). To measure and assess the behaviour of animals, we did not calibrate the three axes of the accelerometer and used one axis to measure the global activity, as the measures on the three axes had a high correlation coefficient (R2 > 0.7). The frequency was set at 0.1 Hz (one measure every ten seconds). Owing to some battery issues, the activity was measured for seven days for the Phurba herd and for five days for the Thaku herd.wGT3X-BT ,13. AcceSpatial associations: Proximities between individuals were measured using Bluetooth technology with the Actigraph wGT3X-BT. A received signal strength indicator (RSSI) [r (RSSI) ,15 was rMatrices of spatial associations were available every day and every night for the duration of the study.GPS locations: The GPS locations of the 12 Phurba herd yaks were scored from March 10 to 24, every minute during the day and every ten minutes during the night. With the help of Dplyr [lubridate [moveHMM [AdehabitatLT [SetNa and Sett0. Animated graphs with maps as the background were obtained by using the R package ggplot2 [gganimate [GGmap [https://doi.org/10.5281/zenodo.7281214, accessed on 4 November 2022).of Dplyr , lubridaubridate , and mov[moveHMM  R packagabitatLT  was used ggplot2 , followeganimate . Two thoe [GGmap  with APIActivity: Kruskal–Wallis tests were used to test the difference in the activity distribution between the two herds and between juveniles and adults in the two herds.Social Network Analyses: Firstly, we calculated the mean number of signals exchanged between loggers during the day and at night for each group and for each cattle . Secondly, the spatial association matrices were uploaded on Gephi 0.92 [phi 0.92 , which wphi 0.92 , which mSpatial cohesion: Using the GPS locations of yaks, after every 30 m we analysed the number of subgroups. We defined a cluster comprised of one or several cohesive individuals (less than 0.5° longitude or latitude of separation) separated from other individuals (or subgroups) by at least 0.5° longitude or latitude.https://doi.org/10.5281/zenodo.7281214, accessed on 4 November 2022.All analyses were made using RStudio 2022.02.2 , α = 0.0Activity: The activity distribution was the same between the two herds  in the Thaku group (mean = 77.5 ± 38.4), indicating that the cattle in the Thaku group were more cohesive than the Phurba (mean = 66.4 ± 32.2) herd. The groups were more cohesive during the night than during the day . In the Thaku group, more signals were received in young individuals due to the tether rope ; groups were more clustered at night than during the day  and the tethering rope increased the modularity . The modularity was significant in the case of the tether rope (Q > 0.03) as the young individuals clustered together. Finally, the mean number of signals received per individual negatively correlated with age, with younger members being closer to other members than old females .Spatial cohesion: The mean number of subgroups observed using GPS was 1.22 ± 0.46. The herd was cohesive (one group) in 78.7% of the scans and two subgroups were observed in 20% of the cases. Three and four subgroups were observed in 1.2% and 0.1% of the scans, respectively. The 24 h period analysis suggested that cohesion varies temporally  but the pairwise comparison tests did not reveal a difference between each hour (p > 0.1). So, a day was divided into six periods of four hours each and the degree of cohesiveness differed during the periods . Higher cohesiveness was observed between 1 a.m. and 4 a.m. than other periods (p < 0.002) except for the period between 5 a.m. and 8 a.m. (p = 0.09). The mean number of clusters between 1 a.m. and 4 a.m. were 1.05 ± 0.23 whilst in other periods the number was around 1.26 ± 0.58. A lower spatial cohesion was observed around 5 p.m. when yaks returned to the village (1.34 ± 0.58).This study is preliminary to deeper research required on the social behaviour of yaks and impact of domestication on this behaviour. This is the first study to highlight the social behaviour in yaks.First, whilst the herds had different herding management, animals in the two herds were cohesive and synchronised their activities. Though the herds were rarely seen on the same pasture, their activity distribution was the same during the day. This distribution was punctuated by the phases of leaving the village  or bothThis study had two limitations: (a) the duration of the study and (b) the number of cattle equipped with loggers. Technological advances can mitigate the constraints met at this altitude that affected data scoring (the extreme cold and lack of electricity affected the batteries). However, this study shows the importance of herd management on yaks’ social behaviour, particularly with the resurgence of the snow leopard ,38 and c


Protein-energy malnutrition and mineral deficiencies in children under five years are major problems in developing countries. The present study was thus carried out with the aim of proposing a weaning flour based on parboiled rice, spirulina, and cashew nut that meets the nutritional needs of children aged 06 to 24 months. To achieve this, the mixture design approach (a 9-point augmented simplex-centroid design) was used to obtain optimal blend of flour. The responses evaluated where proteins, lipids, carbohydrates, minerals , carotenoids, fibers, and ash content. The rheological analyses  were carried out on the optimal flour. It results that the optimal proportion of parboiled rice, spirulina, and cashew nut was, respectively, 85.80, 5.96, and 8.23. These conditions result in a protein, lipids, carbohydrates, carotenoids, ash, fibers, calcium, iron, magnesium, and zinc content of 14.15%, 9.04%, 68.46%, 8.2 mg, 3% ash, 4.98%, 510 mg, 16 mg, 65 mg, and 5 mg, respectively, and an energy value of 411.8 kcal. These optimate conditions resulted also in a peak viscosity of 60 cP and a final viscosity of 86 cP which are lower than the established value (1000 cP). Thus, the weaning flour based on parboiled rice, spirulina, and cashew nut obtained in these optimate conditions can meet the nutritional needs of children aged 06 to 24 months and is therefore an efficient weaning food to fight against child malnutrition. Malnutrition is a major public health problem that affects 821 million people worldwide. In sub-Saharan Africa, 22.8% of children under the age of 5 suffer from this malnutrition  report tNerica L56) was purchased from a farmer in Koutaba, the spirulina from Bol market, and the cashew nut was collected in Garoua. The samples thus collected were forwarded to the laboratory and proceeded into flour and powders for weaning food production.The paddy rice (μm).The paddy rice previously cleaned  was soaked in hot water at 65°C for 6 hours and drained. It was then steamed for 40 min, dried in the shade for one hour, and parboiled for 18 h at 45°C. Once parboiled, the paddy was husked, winnowed, and then milled to obtain flour  was filtered through a very fine sieve and washed to remove impurities. It was then pressed in order to reduce the quantity of water, then spread, and dried at 45°C during 24 hours. Once dried, it was crushed and sifted to obtain a powder of 300 μm.The spirulina collected in Lake Chad basin  to facilitate the dehulling which consists in removing the envelope which surrounded the almond. The almond was then dried, crushed, and sieved at 300 A 9-point augmented simplex-centroid design was applied to obtain the optimal flour. The augmented centered mixing designs explore the entire area of interest of a mixing design and conduct mixing experiments perfectly. Designing was done using Minitab 19.1.0, and the experiments were carried out in triplicate.Three factors were identified: the amount of parboiled rice flour (PRF), the amount of spirulina powder (SP), and the amount of cashew nut flour (CNF). The responses were proteins, lipids, carbohydrates, minerals , carotenoids, fibers, and ash content. The choice of experimental domains was made based on data from the literature review and the chemical composition of each matrix. After choosing the intervals of the different factors, we proceeded to the determination of the number of experiments to be carried according to a three-factor mixture design .Y). The fundamental constraint of the mixtures makes the constant disappear, and the second-degree terms reduce to rectangles. The linear mathematical model of the formulated flour was as follows:The postulated model can be either linear or polynomial of second-degree where the responses are coded by  which must be above 75%, the Absolute Mean Deviation Analysis (AMDA) which must be equal to 0, and the bias factor (Bf) which must be between 0.75 and 1.25 [The validation and strength of an experimental mathematical model require the determination of a number of parameters such as the determination coefficient  (°C)Peak viscosity (PV) (cP)Holding viscosity (HV) (cP)Breakdown (B) (cP)Final Viscosity (FV) (cP)Setback (S) (cP)The pasting properties of optimized flour were characterized by Rapid Viscosity Analysis (RVA). Starch dispersed in water with constant agitation was subjected to a Brabender viscoamylograph which records the evolution of the consistency of the paste during different phases of heating, temperature maintenance, or cooling. The viscoamylograph reflecting the transformations undergone by the starch granules as a function of time during heating shows the evolution of its consistency. To achieve this, 3 g of flour was mixed in 25 ml of distilled water. The resulting suspension was subjected to a progressive heating at the rate of 6°C/min from 50 to 95°C. The constant temperature of 95°C was maintained for 5 min, followed by a cooling of the suspension at the rate of -12°C/min. A new constant 57°C temperature step was maintained for 2 min. Temperatures of the aqueous starch suspension and recorded viscosities (in centipoise or cP) in real time were deduced from the starch weighing parameters. The following viscometric parameters were measured in this work:Graphical representations of the isoresponse curves of the postulated models were performed using Minitab version 19.1.0 software. Data were analyzed with Excel 2013 and Minitab version 19.1.0 software and statistical analyses using SPSS version 22.0 software. The comparison of means was performed by the ANOVA test to classify the treatments in case there was a significant difference. The difference between two data is significant when the actual significance level is below 5%.Y) are expressed as a function of PRF, SP, and CNF which represent the effects of parboiled rice, spirulina, and cashew nut on the response, respectively. The higher the coefficient of these factors, the more the factor influences the response. PRF∗SP represents the interactions of parboiled rice and spirulina on the response, and PRF∗CNF represents the interactions of parboiled rice and cashew nut on the response. The SP∗CNF terms cannot be estimated; so, they were removed from the equation. It shows that cashew nut flour is the factor that contributes the most to the equations for proteins, carbohydrates, ash, iron, calcium, zinc, magnesium, phosphorus, and carotenoids. The factor that influences the most potassium, sodium, fibers, and lipids is spirulina. Cashew nut flour contains various factors such as tannins, phytic acid, amylase inhibitors, and trypsin inhibitors that limit the absorption of carbohydrates, proteins, and minerals [The following regression equations were obtained after analysis of the different responses . These responses Proteins:(ii)Lipids:(iii)Carbohydrates:(iv)Ash:(v)Fibers:(vi)Iron:(vii)Calcium:(viii)Sodium:(ix)Zinc:(x)Magnesium:(xi)Potassium(xii)Phosphorus(xiii)Carotenoids:Here are the following factors:R2), the AAMD , and the Bf (bias factor) obtained for the analysis of the different responses. It appears that the R2 of the different responses is greater than 75%, the AADM is equal to 0, and the Bf is between 0.75 and 1.25. Indeed, when these three coefficients respected standard values, we consider that there is an adequacy between the experimental responses and the predicted responses given by the model [he model .p < 0.05) between the responses given by the model and those obtained in the laboratory, and the desirability varies from 0.92 to 1. Thus, our optimal proportions of parboiled rice flour, spirulina, and cashew nut flour are validated.After the analysis of the different responses, a compromise was made between the different optimal conditions obtained from the responses obtain by the different tests. The rectangle of β-carotene can be metabolized into vitamin A [β-carotene, β-cryptoxanthin, α-carotene), as they contribute to their intake and also have a great antioxidant capacity. They can inhibit the propagation of lipid peroxidation and decrease oxidative damage to DNA, thus protecting against cancer. They are also very important as immunomodulators and in cellular communication [The mineral content is mainly influenced by the amount of spirulina and cashew nut in the formulated flour. The optimal values obtained for iron, calcium, sodium, zinc, magnesium, potassium, and phosphorus are, respectively, 16 mg, 510 mg, 123 mg, 5 mg, 65 mg, 581,8 mg, and 282,5 mg, per 100 g of dry matter. The amounts of iron, potassium, phosphorus, and sodium are in accordance with the recommended values . Howeveritamin A . Carotennication .Energy value of the optimized weaning flour is within the value recommended by  for chilSubsequently, an increase in viscosity is observed with increasing temperature until the maximum peak (peak viscosity). This peak viscosity indicates the thickening and water holding capacity of the starch . Thus, tThe peak viscosity of the optimal flour obtained can be further explained by its low amylose content (19.75 g), the particle size of the starch source (parboiled rice flour), and amylose-proteins and amylose-lipid interactions . This isBreakdown and holding viscosity refer to the degree to which the viscosity of starches drops after gelatinization . It can Then, a gradual increase in viscosity to final viscosity is observed. This is reflected in the ability of the starch to retrograde upon lowering the temperature due to molecular rearrangement and the formation of amylose-lipid complexes . Thus, tThe difference between the final viscosity and the holding viscosity represents the ability of the starch to retrograde. According to , this isThe setback of the optimal formulated flour is lower than that obtained by . This diThe analyses carried out show that the different samples used for the formulation through the mixing plan allowed to find the optimal conditions. Thus, to have a good weaning food of nutritional quality meeting the standards, 85.80% of parboiled rice flour, 5.96% of spirulina, and 8.23% of cashew nut are required. The final viscosity obtained for our flour is lower than that established by the standard and therefor meets the nutritional needs of children in fewer meals, thus allowing a more effective fight against protein-energy malnutrition and hidden hunger. In view of these results, the formulation of a weaning food based on parboiled rice flour, spirulina, and cashew nut enables us to have a weaning food of good nutritional value and acceptable rheological properties. This study also provides alternatives to the weaning foods in Chadian market as well as in the subregion. The addition of spirulina and cashew nut enriched the rice in the required contents in proteins, lipids, minerals, and carotenoids.