These heaviest manganates crystallise as polymeric zig‐zag chains propagated by AM⋅⋅⋅π‐arene interactions. Key to their preparation is to avoid Lewis base donor solvents. In contrast, using multidentate nitrogen donors encourages ligand scrambling leading to redistribution of these bimetallic manganate compounds into their corresponding homometallic species as witnessed for the complete Li ‐ Cs series. Adding to the few known crystallographically characterised unsolvated and solvated rubidium and caesium s‐block metal amides, six new derivatives ([{AM(N‘Ar)}∞], [{AM(N‘Ar)⋅TMEDA}∞], and [{AM(N‘Ar)⋅PMDETA}∞] where AM=Rb or Cs) have been structurally authenticated. Utilising monodentate diethyl ether as a donor, it was also possible to isolate and crystallographically characterise sodium manganate [(Et2O)2Na(nBu)Mn[(N‘Ar)2], a monomeric, dinuclear structure prevented from aggregating by two blocking ether ligands bound to sodium.Rare examples of heavier alkali metal manganates [{(AM)Mn when donor solvents are absent but introducing the common donors TMEDA or PMDETA leads to breakdown of the ates together with ligand exchange, which results in the formation of new solvated heavier alkali metal amides and alkylmanganese amides. Manganese occupies a unique position in the centre of the periodic table as it exists in oxidation states ranging from −3 to +7,2;[3]−,4]2−.[2Li2Mn(CH2SiMe3)4] to promote oxidative homocoupling of aryliodides.The first applications of organomanganese(II) rest on the pioneering reports by Normant and Cahiez in the 1970s.2;10, 11manganation (AMMMn),3)2}3Li(THF)]2Mg2(μ‐H)2{N(iPr)2}4(toluene)2]2Mn2(μ‐H)2{N(iPr)2}4(toluene)2]2  that form effective catalysts for hydrogenation of olefins.2]2, which was first reported and structurally characterised by Hill.Though not studied in much detail, AMMWe would advocate that it is now considered vital to include the whole of group 1 (Li ‐ Cs) when investigating catalytic performance, since gradations in reactivity can take place depending on which alkali metal is used. Investigations to gain more structural insights in whole series of group 1 complexes often exclude the heavier homologues due to the synthetic challenge or inaccessibility,42, 43+ is commonly found as a counter‐cation in water soluble coordination compounds or caged in cluster complexes containing manganese in the oxidation state +2 or +3,Investigation and deeper insight into the class of Mn(II) compounds remains challenging, since stringent exclusion of air to prevent unwanted oxidation to Mn(IV) species is paramount, while characterisation by NMR spectroscopy is hindered by the paramagnetism of Mn(II). Additionally, isolation and handling of the mediating alkali metal alkyl precursor is impeded with increasing electropositivity of the group 1 metal (Cs>Rb>K>Na>Li), as has recently been shown in the challenging structural elucidation of the heaviest alkali‐metal‐benzyl complexes by Strohmann.2O, TMEDA  and PMDETA  in the success or failure to isolate challenging manganate‐complexes instead of a mixture of their heteroleptic, monometallic alkyl‐amido Mn(II) compounds and alkali metal amides, derived from manganate‐complex destruction. The heaviest homologues of these alkali metal amides are also discussed extensively in terms of their structural diversity when solvated with said donors. We also reveal how exclusion of donor solvents allowed the successful synthesis and structural characterisation of the, to the best of our knowledge, first Rb‐manganate with manganese solely in the oxidation state +2 and its heavier Cs‐homologue prepared in hydrocarbon solvents.With this background, this study sets out to merge these different aspects by attempting to synthesise and characterise a series of new alkali metal manganates, with particular emphasis on rubidium and caesium examples. First, we highlight the influence of the coordinating donor ligands, THF, Et‘Ar)2‘Ar=N(SiMe3)(Dipp), where Dipp=2,6‐iPr2‐C6H3] and commercially available n‐butyl lithium solution in hexane. Unfortunately, these co‐complexation attempts, that is the formal addition of two monometallic species to yield a new bimetallic complex, repeatedly resulted in the formation of mixtures of lithium amide LiN‘Ar or its monometallic coordination compounds with various donor solvents and yellowish residue, which failed to deposit any crystalline material for further investigation 2,‘Ar ⋅ (THF)3,‘Ar ⋅ (TMEDA)‘Ar ⋅ (PMDETA)2O, THF, TMEDA and PMDETA respectively, consistent at least in part with manganese‐lithium exchange phenomena. The total absence of additional donor solvent promoted the formation of formal twofold ligand exchange to yield one equivalent of the [LiN‘Ar]22SiMe3)2}∞],[2SiMe3)2}∞].Changing the lithium alkyl precursor to the neosilyl lithium LiCH+ cation preferring the harder nitrogen of the amide ligand, we next investigated the influence of heavier alkali metals on this metathesis reaction with respect to the donor solvents applied. Treatment of Mn(N‘Ar)2 dissolved in benzene with freshly prepared nBuNa under reflux conditions leads to precipitation of a white solid of unknown composition. Attempts to recrystallize this material in aromatic solvents failed, but slow diffusion of n‐hexane into a concentrated Et2O solution yielded colourless crystals in 71 % yield and suitable for X‐ray crystallographic determination, revealing the bimetallic sodium manganate [(Et2O)2Na(nBu)Mn[(N‘Ar)2] (1) . However, Evans methods could be used to assess its magnetic moment (effective magnetic moment μeff=5.94 μB) which is consistent with five unpaired electrons in a high‐spin state for the Mn(II) centre. Manganate 1 crystallised in the monoclinic primitive space group P21/c and can be classified as a contacted ion pair type complex, containing a 2 : 1 amido/butyl stoichiometric ratio, similar to that of previously reported Mn(II) synergistic metalating agents.. This three‐coordinate Mn moiety is connected through two bridges to the Na1 centre, which overcomes intermolecular bonding tendencies by coordination to two diethyl ether molecules. One bridge consists of the α‐carbon of the bridging butyl ligand and the other is the η3‐coordination (Na1‐C25: 3.1190(14) Å, Na1‐C26: 2.8122(14) Å, Na1‐C27: 3.0180(13) Å) to one of the amido ligand's Dipp groups. These alkali metal‐arene interactions are decisive for the formation of the polymeric helical chain structure in the related magnesium compound [{(Na)Mg(nBu)(N‘Ar)2}∞]Compound It should be noted that adding the more strongly donating oxygen donor solvent THF did not allow for isolation of a defined species. This has been the incentive to investigate for possibly complex formation tendencies with multidentate donor ligands.‘Ar)⋅PMDETA. While the Mn counterpart could not be isolated, it can be assumed that Mn(N‘Ar)(nBu)⋅PMDETA was also formed, though this species appears to be highly soluble in hydrocarbon solvents and attempts to isolate it as a crystalline solid were unsuccessful. This donor‐mediated dissociation process has already been observed in related magnesiate compounds.Switching to the tridentate nitrogen donor PMDETA led to complex dissociation with the isolation of single metal Na(N Mn(N‘Ar)nBu⋅PMDEnBu)(N‘Ar)2] in TMEDA/n‐hexane mixtures gave two distinct sets of crystals, suitable for X‐ray crystallographic characterisation, which were identified as monometallic complexes [Na(N‘Ar)⋅TMEDA]2‘Ar)(nBu)⋅TMEDA (2 a) MnnBu species containing non‐stabilised n‐butyl ligands remain scarce in literature, and free amine H‐N‘Ar with one equivalent of TMEDA led to intractable product mixtures.Isolation of neutral manganese complex terature,14, 802 a displays a distorted‐tetrahedral coordination geometry in the solid state, which is typical for four‐coordinate Mn(II) complexes 2⋅TMEDA2⋅TMEDA2⋅TMEDA2 a is 104.81°. This distortion from an ideal tetrahedral geometry is pronounced by the widening of the amido‐metal‐alkyl bite angle [N1‐Mn1‐C1: 124.80(6)°], which emphasises the steric hindrance applied by the aryl‐silyl‐amide ligand.Mn1 forms short bonds to the anionic α‐carbon of the butyl ligand [Mn1‐C1: 2.1479(17) Å] and the amido‐nitrogen [Mn1‐N1: 2.0741(12) Å] and exhibits longer dative bonds to the TMEDA nitrogen atoms . The metal‐TMEDA bite angle (N2‐Mn1‐N3) of 79.96(4)° is somewhat comparable to those of previously reported TMEDA‐ligated manganese(II) complexes, for example Mn(CH2SiMe3)2SiMe3) reacts at room temperature with Mn(N‘Ar)2 to afford the co‐complexed products as a white precipitate from a yellow benzene reaction mixture. However, recrystallisation from a TMEDA\n‐hexane solution leads repeatedly to ligand scrambling to yield the monometallic complexes Mn(N‘Ar)(CH2SiMe3)⋅TMEDA (2 b) and the corresponding alkali metal amides AM(N‘Ar)⋅TMEDA. The lighter sodium and potassium homologues have already been structurally elucidated in the literature [AM=Na: dimeric [Na(N‘Ar)⋅TMEDA]2,‘Ar)⋅TMEDA}∞,‘Ar)⋅TMEDA}∞, AM=Rb (3‐Rb⋅TMEDA) and AM=Cs (3‐Cs⋅TMEDA) has been collected to this point  r2 b  Å] bond and Mn1‐N1 [2.0623(15) Å] bond compare favourably to the structurally similar complex 2 a, with the main difference lying in the wider N1‐Mn1‐C1 bite angle of 135.19(7)° in the neosilyl‐substituted complex 2 b, which is a consequence of the increased steric bulk induced by the CH2SiMe3 group.Determined by X‐ray crystallography, the molecular structure of ‘Ar)(CH2SiMe3)⋅PMDETA species and homometallic AM(N‘Ar)⋅PMDETA (AM=Na: Na(N‘Ar)⋅PMDETA,‘Ar)⋅PMDETA}2],‘Ar)⋅PMDETA}∞] (3‐Rb⋅PMDETA), AM=Cs: [{Cs(N‘Ar)⋅PMDETA}∞] (3‐Cs⋅PMDETA)) respectively.In accordance with previous observations regarding ligand scrambling, carrying out this set of co‐complexation reactions in the presence of tridentate PMDETA, instead of TMEDA, leads to formation of a yet unidentified Mn(N‘Ar in benzene solution was heated to reflux in a transamination reaction to yield the desired donor‐free [{Rb(N‘Ar)}∞] (3‐Rb) and [{Cs(N‘Ar)}∞] (3‐Cs), which can be crystallised and isolated from the reaction mixture in 82 % and 75 % yield, respectively  with one equivalent of H‐N3‐AM could be identified by X‐ray crystallography, as well as by one‐ and two‐dimensional NMR and infrared spectroscopy . 3‐Rb crystalises in the monoclinic space group P21/c and 3‐Cs in the tetragonal space group P‐421m while both complexes adopt a polymeric chain‐type structure interconnected by attractive intermolecular anagostic interactions in the solid state  ‐ 3.321(2) Å] and anagostic Rb⋅⋅⋅C(H3)SiMe2 interactions [Rb⋅⋅⋅C14’=3.674(3) Å]. Neighbouring chains link along the b‐axis through a second anagostic Rb⋅⋅⋅C(H3)SiMe2 interaction [Rb1⋅⋅⋅C15’’’=3.486(2) Å]. The strongest metal bond in 3‐Rb is the mostly ionic metal amide bond [Rb1‐N1=2.8322(15) Å], which completes the coordination sphere around the rubidium metal centre leading to a much wider ipso‐carbon‐nitrogen‐silicon angle [C1‐N1‐Si1=140.88(13)°] in the amide ligand compared to lighter sodium  and potassium  homologues. The heaviest group 1 amide 3‐Cs has strikingly different structural properties as the packing in this ionic compound renders the Cs−N distances too long to be considered a chemical bond [Cs1⋅⋅⋅N1=3.725(1) Å and Cs1⋅⋅⋅N1’=4.326(1) Å]. While the crystal structure can still be described as that of a polymeric chain structure, the propagation alongside the crystallographic c‐axis proceeds through a second Cs⋅⋅⋅π‐arene interaction towards the Dipp group of the next asymmetric unit with both aryl‐rings being coordinated in a η6‐fashion . This energetically favourable π‐stabilisation in addition to two intermolecular anagostic Cs⋅⋅⋅C(H3)SiMe2 interactions [Cs1⋅⋅⋅C9’’’=Cs1⋅⋅⋅C9’’’’=3.661(1) Å] and intramolecular longer Cs⋅⋅⋅C(H3)SiMe2 contacts towards the isopropyl moieties [Cs1⋅⋅⋅C7=3.679(1) Å] fill the coordination sphere around the large Cs metal. With the metal being more separated from the amide N atom, the corresponding ipso‐carbon‐nitrogen‐silicon angle in the amide ligand in 3‐Cs is widened significantly [C1‐N1‐Si1=148.8(5)°]. In comparison to the vast body of well‐studied lithium amides in the literature, the number of crystallographically characterised polymeric sodium and potassium amides are limitedFor rubidium amide 3‐AM in hexane and subsequent addition of chelating TMEDA led to transparent solutions, which yielded colourless crystals after storage at −20 °C. X‐ray crystallography and one‐ and two‐dimensional NMR spectroscopy confirmed the same products 3‐AM⋅TMEDA, as have been found in the corresponding dissociation of the co‐complexed manganate. In contrast to the lighter Li to K congeners, 3‐Rb⋅TMEDA and 3‐Cs⋅TMEDA are not deaggregated by the donor solvent, but a polymeric chain structure is retained  moiety. In contrast to parent amide 3‐Rb, the absence of interconnecting anagostic contacts leads to an infinite zig‐zag chain in 3‐Rb⋅TMEDA that propagates parallel to the crystallographic b‐axis through one η6‐coordinating Rb⋅⋅⋅π‐arene interaction  and one η4‐coordinating Rb⋅⋅⋅π‐arene interaction . In addition to the metal‐ π interactions the metal's coordination sphere is filled by the two dative Rb nitrogen bonds from TMEDA  leading to the absence of an explicit alkali metal amide bond comparable to the structural motif in the heavier donor‐free complex 3‐Cs. The distances of the rubidium metal to the amide nitrogen atoms in 3‐Rb⋅TMEDA [Rb1‐N1=4.4575(17) Å and Rb1‐N1’=4.0571(17) Å] are too long to be considered a chemical bond, rendering the Dipp‐N‐(SiMe3) moiety to an almost linear geometry [C1‐N1‐Si1=174.41(16)°], which emphasizes the strong tendencies for the soft alkali metal to predominantly bind to softer ligand moieties. Similar to its lighter rubidium homologue the TMEDA complex 3‐Cs⋅TMEDA crystallises in a monoclinic space group and forms linear but twisted chain structures that propagate alongside the crystallographic a‐axis. However, in contrast to 3‐Rb⋅TMEDA, there are two similar but crystallographically independent Cs environments. While each metal coordination site is built up by two dative metal nitrogen bonds [mean: Cs‐NTMEDA=3.27 Å] towards TMEDA, only one η6‐coordinating Cs⋅⋅⋅π‐arene interaction  towards the Dipp group of the amide is formed; whereas a strong metal amide bond  together with weaker Cs+⋅⋅⋅Meδ−‐Siδ+ electrostatic interactions is present at both Cs sites. The Cs coordination to the amide nitrogen consequently narrows the Dipp‐N‐(SiMe3) angle [C1‐N1‐Si1=135.5(5)° and C25‐N2‐Si2=138.4(6)°] for both coordination sites in 3‐Cs⋅TMEDA, so that the SiMe3 groups exhibit a syn conformation within the chain structure.The crystallographic determination of ‘Ar) (AM=Li−K) can be similarly deaggregated by adding tridentate PMDETA. In the case of Li(N‘Ar)⋅PMDETA and Na(N‘Ar)⋅PMDETA this deaggregation takes place all the way down to the monomers; whereas the heavier potassium amide PMDETA adduct [{K(N‘Ar)⋅PMDETA}2] forms a dimer in the solid state.3‐AM  in benzene or hexane treated with PMDETA yielded clear solutions and storing mixtures of 3‐AM in hexane/PMDETA at −20 °C yielded colourless crystals of the corresponding alkali metal amide donor adduct complexes [{AM(N‘Ar)⋅PMDETA}∞] . The compounds could be structurally identified by X‐ray crystallography and one‐ and two‐dimensional NMR spectroscopy, confirming the previous findings from the complex destruction pathway in the corresponding bimetallic manganate complexes  Å and Rb2‐N5=2.882(2) Å; Cs1‐N1=3.036(4) Å] and three shorter dative metal‐nitrogen bonds from the PMDETA chelating ligand  with the alkali metal being additionally capped by the neighbouring Dipp moiety via η2‐coordination to the arene ring in 3‐Rb⋅PMDETA and with a η1‐coordination in the heavier caesium homologue. Encapsulation of the alkali metal in the amide/PMDETA donor pocket in combination with different crystal packing in 3‐AM⋅PMDETA compared to the packing observed in 3‐Cs and 3‐Rb⋅TMEDA  angles C1‐N1‐Si1 [mean: 146.5(2)° in 3‐Rb⋅PMDETA and 152.8(4)° in 3‐Cs⋅PMDETA] in the amide ligand that lie in between that of the parent amides 3‐AM and 3‐Rb⋅TMEDA.Unlike in 2SiMe3)(N‘Ar)2}∞] , which precipitate from benzene reaction mixtures and can be isolated in reasonable yields of 81 % (AM=K), 52 % (AM=Rb), and 66 % (AM=Cs), respectively MnMn(CH2SiMe3)(N‘Ar)2}∞] (4‐Rb) and isostructural [{(Cs)Mn(CH2SiMe3)(N‘Ar)2}∞] (4‐Cs) amenable to X‐ray crystallographic determination . Attempts to record meaningful NMR spectra in benzene‐6d and THF‐8d were unsuccessful due to the paramagnetic nature of Mn(II). A solution state magnetic measurement of 4‐AM via the Evans method gave μeff values  that lie within the range of the expected spin‐only value for a high spin MnII‐d5 configuration (μS.O.=5.92 μB).Recrystallisation from toluene solutions via slow diffusion‐layering with 4‐Rb and 4‐Cs crystallise in the monoclinic primitive space groups P21\c and P21\n, respectively. Both compounds form infinite zig‐zag type chain structures, in which the tris‐coordinate manganate centres are interconnected via group 1 metals engaging in stabilizing AM⋅⋅⋅π‐arene interactions. Each alkali metal is coordinatively satisfied by interaction with the alkyl ligand's alpha carbon atom , with one η6‐coordination to one aryl unit within the asymmetric unit and one η6‐coordination with an aryl unit within the neighbouring unit, thus propagating through the crystallographic c‐axis in the case of 4‐Rb and the crystallographic b‐axis for 4‐Cs. The coordination environment surrounding the Mn(II) centre is best described as distorted trigonal planar with the sum of bond angles around the Mn centre being 359.32° in 4‐Rb and 359.79° in 4‐Cs. The mean Mn amide bond lengths  and manganese carbon bond length  match in dimensions, not only showing the structural similarity of the manganate core within the two complexes, but also being consistent with the fact that the group 1 metal and the manganate ion are predominately electrostatic in nature. A possible flexibility of the system regarding the compensation of the group 1 metal's size is exerted by rotation of one silyl group. In 4‐Rb all three silyl moieties are orientated in a facial way allowing for a shorter intermolecular anagostic Rb1⋅⋅⋅C(H3)SiMe2 contact [Rb1‐C20=3.307(6) Å], while 4‐Cs exhibits one longer and one shorter anagostic Cs1⋅⋅⋅HCH2SiMe2 contact [Cs1⋅⋅⋅C21’=4.103(5) Å and Cs1⋅⋅⋅C22’=3.699(4) Å]. These AM+1⋅⋅⋅Meδ−‐Siδ+ electrostatic interactions are commonly observed in s‐block chemistry in generalThe heavier group 1 metal manganate complexes 4‐AM  in the aromatic solvents benzene or toluene to the coordinating oxygen donors Et2O or THF did not yield crystalline material suitable for X‐ray crystallographic analysis, which may be explained by a lower tendency of these non‐chelating ligands to disintegrate the bimetallic species. In contrast to previously reported Mn(II) metallators4‐AM did not show any signs of direct manganation or group 1 arene metallation reactions, which emphasises the importance of template effects that play a crucial role in AMMMn reactions.Exposure of manganates ‘Ar)2 with alkyl lithium reagents give reaction mixtures with limited stability under investigated conditions and exclusively led to twofold ligand exchanged monometallic lithium amide and bis‐alkyl‐manganese compounds. Addition of the polydentate coordinating solvents TMEDA and PMDETA allowed for more detailed structural identification of heteroleptic, monometallic alkyl‐amido Mn(II) complexes 2 a and 2 b which were crystallographically characterised, as well as the monometallic alkali metal amide complexes 3‐AM⋅TMEDA and 3‐AM⋅PMDETA , which in contrast to their lighter homologues adopt infinite linear or zig‐zag arrangements as identified in their solid state structures. Structural diversity in these adduct‐complexes and in the parent, donor‐free alkali metal amide compounds 3‐Rb and 3‐Cs, obtained from a transamination reaction, is discussed, emphasising the complexity in seemingly simple alkali metal amide systems. The co‐complexation of butyl‐sodium with said Mn(II) amide yields monomeric, bimetallic sodium manganate 1 in diethyl ether, a potential metalating agent in AMMMn chemistry, which will be targeted in future work. The first structurally characterised Rb‐manganate 4‐Rb with manganese solely in the oxidation state +2 is reported, together with its heavier Cs‐homologue 4‐Cs, which represents the first example of a mixed amido‐alkyl caesium manganate. Presumably the AM‐arene interactions of the heavier AM metals Rb and Cs favour formation of the bimetallic species, contrary to the lighter Li and Na homologues in which these arene interactions are less pronounced, which leads to ligand redistribution reactions. Likewise, adding the donor solvents TMEDA and PMDETA may block the coordination sites of the AM metals, which competes with the AM‐arene interactions, thus favouring AM cleavage and complex destruction.In conclusion, we have shown that treatment of literature known manganese‐bisamide Mn the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Number(s) The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The impact of the Coronavirus disease 2019 (COVID-19) pandemic was more pronounced on the well-being of persons with disabilities, especially in low- and middle-income countries. There is documented evidence of the rippling effects of COVID-19 on persons with disabilities. However, not much is known about the impact of COVID-19 on the rehabilitation of persons with aphasia.The scoping review explores how COVID-19 affected the rehabilitation of persons living with aphasia.A scoping review was conducted using Arksey and O’Malley’s framework. A search was conducted on Science Direct, PubMed, Medline, Scopus, ProQuest and Google Scholar, to identify relevant studies published between 2019 and 2022. Data were analysed using thematic analysis.Most studies regarding the effects of COVID-19 on persons living with aphasia were conducted in the United Kingdom. Five themes emerged from the data, namely, (1) negative impact on rehabilitative care, (2) telehealth and its limitations, (3) impact on social participation, (4) compromised caregiver involvement and (5) mental health challenges.Findings highlight the need for healthcare professionals to pursue innovative ways in which aphasia rehabilitation and conversational support programmes can be made accessible to persons with aphasia, despite the limitations brought about by a pandemic. Telerehabilitation programmes need to be tailored to the needs of persons with aphasia if they are to be successful. This study highlights the importance and need for the prioritisation of mental health services for persons with aphasia and their caregivers during a pandemic. Globally, stroke is a leading cause of mortality and disability  because when communication is affected, vocational, family as well as social relations are affected, often leading to frustration, isolation, hopelessness and depression , reference number: STA_2021_41. The articles accessed and reviewed are in the public domain and thus the authors did not need permission to conduct the review. This study did, however, follow all ethical standards for a research without direct contact with human or animal subjects.n = 1), whilst the mixed methods design consisted of two surveys conducted at two different periods. The first survey was with (n = 544) speech language therapists whilst the second was with (n = 413) speech language therapists. Geographically, three (43%) of the studies were conducted solely or in collaboration with researchers from the United Kingdom, 29% (n = 2) were situated in the USA, 14% (n = 1) in Italy and 14% (n = 1) in Malaysia.Methodologies used in the seven studies included one qualitative (14%), one mixed methods study (14%), one case presentation (14%) and four opinion and position statements (58%). The qualitative design consisted of one single-subject design , who propose that before assigning patients to telerehabilitation, therapists need to be aware of factors such as their communication and motor abilities, severity, chronicity and symptoms of aphasia. A study conducted by Neate, Kladouchou, Wilson and Shams  agrees wShafer, Shafer and Haley , p. 635 Findings from the study suggest the need for rehabilitation healthcare professionals to pursue innovative ways in which aphasia rehabilitation support and conversational programmes can still be made accessible to persons with aphasia despite the limitations brought about by COVID-19 or any other pandemic or circumstance that may hinder face-to-face patient contact. Group rehabilitation therapy is important for facilitating conversations as well as overcoming conversational barriers that persons with aphasia and their families experience. Conversational groups create a social interactive environment which is ideal for practising communication and participation, which are good for the emotional and mental well-being of persons with aphasia and their families.Telerehabilitation provides an alternative means to continue patient rehabilitation care, including online aphasia intervention and conversational groups. However, in order for telerehabilitation to be successful, special consideration need to be made for the physical and communicational needs of the person with aphasia and their family. The authors would go as far as to argue for contextual relevant consideration such as data costs, connectivity challenges because of poor infrastructure, health and digital literacy, which are mostly experienced in LMICs. The above-mentioned factors mostly experienced in LMICs may not have come through in the results of this study because of the paucity of literature of the subject as it pertains to aphasia rehabilitation during the pandemic. However, they are a reality in these contexts, and they have the potential to impede the delivery of rehabilitation services. Studies on the subject from LMICs are needed so as to shed some light onto the current telerehabilitation trends when managing persons with aphasia so that context-specific solutions can be forged.As the global need for telerehabilitation continues to increase, it is therefore necessary to have a rehabilitation healthcare workforce that possess the necessary knowledge, skills and experience, in telerehabilitation. There is a need for renewed and deliberate efforts to integrate telerehabilitation into the academic curriculum for current and future rehabilitation healthcare students. Current rehabilitation healthcare professionals can enhance their telerehabilitation skills through continued professional development activities.


Their cell viability demonstrated that twelve compounds exhibited appreciable potencies against NAFLD . Furthermore, the most potent compound 5f effectively prevented NAFLD progression as evidenced by the change in histological features. 5f significantly reduced total cholesterol and triglyceride levels in vitro/in vivo, and the effects of 5f were significantly stronger than those of the control drug. The proteomic data showed that oxidoreductase activity was the most significantly enriched, and this finding was consistent with docking results. In summary, this validated presynthesis prediction approach was cost-saving and worthy of popularization. The novel synthetic phenyl ketone derivative 5f holds great therapeutic potential by modulating oxidoreductase activity to counter NAFLD.Nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions with no pharmacological treatment approved. Several highly accessible computational tools were employed to predict the activities of twelve novel compounds prior to actual chemical synthesis. We began our work by designing two or three hydroxyl groups appended to the phenyl ketone core, followed by prediction of drug-likeness and targets. Most predicted targets for each compound overlapped with NAFLD targets (≥80%). Enrichment analysis showed that these compounds might regulate oxidoreductase activity. Then, these compounds were synthesized and confirmed by IR, MS,  In the current era of big data, computational methods have led to a more cost-, time-, and labor-efficient drug discovery process . Among cNonalcoholic fatty liver disease (NAFLD) encompasses a disease spectrum, from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. The prevalence of NAFLD is estimated to be 25% globally and continues to increase . Unfortu1H, and 13C nuclear magnetic resonance (NMR) were used to confirm the structures. Thirdly, cell viability experiments were conducted to validate the computational prediction results initially. Another validation was made by evaluating the effects of the most potent compound 5f on in vitro/in vivo NAFLD models. The proteomic study was performed to search for a mechanism by which 5f intervention may be affecting NAFLD and to validate prediction results further. Finally, molecular docking was conducted to help understand the mechanism of 5f.To our knowledge, this was the first time to apply computational technologies to predict the drug-likeness and biological activities of novel phenyl ketone derivatives for treating NAFLD prior to chemical synthesis. The prediction results were in line with subsequent experiment results. The design of this study is shown in p) ≤ 5 [2 and number of rotatable bonds (NRB) ≤ 10.Preevaluation of drug-likeness is valuable during the early stage of drug discovery. Compounds with desirable bioavailability can be preselected in a time-efficient manner . Compounp) ≤ 5 . Anotherp) ≤ 5 , which sp, which was a vital consideration for suitable lipid solubility for the drugs to penetrate through biomembranes. The presence of HBAs and HBDs in the molecular structure of a solute favors aqueous solvation and tends to make the solute less lipophilic [2, suggesting good bioavailability by oral route [The twelve designed compounds in our lab were checked for the drug-likeness features . MW was pophilic . HBAs anal route . Moleculal route .in silico ligand-based target prediction is regarded as an effective tool and has shown high-quality performance [X-axis. We found that most predicted targets (≥80%) of each compound overlapped with the molecular targets of NAFLD, with some  even reaching 90%.A fundamental step in the discovery context is to predict the targets of a given drug-like compound (ligand) and investigate its potential pharmacological effects. To achieve this process, formance . The ligformance . Based oformance , DIGEP-Pformance , and Tarformance , were ap5a-l is outlined in 2a-l were prepared by treating 1a-l with commercially available dimethylformamide (DMF) in SOCl2. Subsequent Friedel–Crafts' acylation provided the second benzene ring via AlCl3-catalyzing. 2a-l immediately reacted with electron-donating substituted aryl rings such as 1,2-dimethoxy-benzene and 1,2,3-trimethoxy-benzene to afford corresponding 3a-l. Further, the resulting 3a-l underwent halogenation with SO2Cl2 or N-bromosuccinimide (NBS) to give 4a-l. Finally, 4a-l were demethylated with BBr3, leaving the hydroxyl groups exposed. Formation of desired compounds 5a-l was achieved.Encouraged by the above prediction results, all twelve compounds were selected for the chemical synthesis. Overall synthetic route of phenyl ketone derivatives in vitro. HepG2 cells were treated with 500 μM palmitic acid (PA) for 24 hours [50) values. As shown in 50values ≤ 13.5 μM), which initially verified the results of our computational prediction. We found that three butyl-substitution groups on phenyl ketone bearing analogues resulted in different potencies. The isobutyl group substitutions  showed the strongest potency, while the n-butyl substitutions  and tert-butyl  caused lower potency. This trend was in agreement with the percentage of overlapping targets listed in A widely accepted modeling condition was used to resemble the NAFLD condition 24 hours , 30. The5f (EC50 = 10.2 nM), was further evaluated for the activities to reduce triglycerides (TG) and total cholesterol (TC) levels. As presented in Figures p < 0.01). Treatment of HepG2 with various dose levels of 5f decreased the TG accumulation and TC content compared with the model group (p < 0.05 or p < 0.01). Silybin (sil), a polyphenolic flavonoid, had been used for a long time to treat acute and chronic liver diseases [μM sil treatment, 10 μM and 25 μM 5f treatment showed no statistical differences, implying that 5f may have a lower effective dose.The most promising compound, diseases . It was 5f on NAFLD was further investigated with a murine model of a high-fat diet (HFD) induced [p < 0.05). Treatment with 5f (12.6 mg/kg or 25 mg/kg) resulted in significant weight loss (p < 0.05). Consistent with these observations, serum TC and TG contents  compared with HFD-fed group, confirming the effects of 5fin vivo. Regarding body weight, serum TC and TG levels, 12.6 mg/kg and 25 mg/kg 5f treatment showed no statistical differences compared to 40 mg/kg sil treatment.The effect of compound  induced . Mice fe induced . As show Figures  remarkab5f-treated mice in comparison to the HFD-fed mice. Results of animal experiments were consistent with in vitro experiments that supported the hypothesis that 5f had promising therapeutic potential against NAFLD.As the “gold standard” for assessing hepatic damage is liver biopsy, liver sections were used for hematoxylin and eosin (H&E) staining. As shown in μM compound 5f intervention group through in vitro experiments. Totally, 290 DEPs were identified . GO analysis was then carried out, and the top fifteen molecular function terms are listed in 5f intervention, suggesting the activity of 5f might be mediated by oxidoreductase activity. The term “oxidoreductase activity” was also the top term of enrichment analysis results in To further test this hypothesis, we used tandem mass tag-based quantitative proteomics to identify differentially expressed proteins (DEPs) between the PA-treated group and 25 p value. The two most significant DEPs were NQO1 and MTRR, which were two flavoreductases. NQO1 (NADPH quinone oxidoreductase 1) can catalyze quinone through a one-step two-electron reduction process, scavenging superoxide and protecting endogenous antioxidants. It is well known that NQO1 and MTRR are related [As can be seen in  related . The MTR related . Further related . The hep related . Six DEG related . NDUFB6  related .5f was docked into the nineteen DEPs clustered in the term “oxidoreductase activity,” and the 3D structures for the other six DEPs were not available. Like the above-mentioned experiments on cells and mice, sil was used as the reference compound to perform docking. A docking is considered successful if the root-mean-square deviation (RMSD) is less than or equal to 2.0 [5f into nineteen DEPs. Hollow circles indicated RMSD values for docking of sil into nineteen DEPs. All dockings were within an RMSD threshold of 2.0, and this ensured the docking accuracy. According to the rule of SYBYL software, the total score shows the binding affinity between the drug and target. A higher total score suggests a closer interaction between drug and potential target. When total score is ≥4, the drug is considered a candidate to be studied continuously [5f with nineteen DEPs were indicated with filled triangles in p values in 5f scored higher than sil against most DEPs. The binding of NQO1 with 5f yielded the highest total score value, and the visualization is shown in Figures 5f was connected to NQO1 through six hydrogen bonds, suggesting that 5f was very likely an excellent substrate for NQO1. Similar to previous studies [5f could also bind firmly to the TRP105 residue by hydrogen bonding interaction. To sum up, our proteomics analysis indicated that compound 5f could exert promising therapeutic potential against NAFLD via modulating oxidoreductase activity.Computational methods like molecular docking provide a necessarily complementary way to experimental methods to predict molecular interactions and explore binding modes of small molecules with targets . Compounl to 2.0 . The detinuously . The obt studies –48, 5f c5f with dual lipid/weight-lowering properties through modulating oxidoreductase activity as a potential therapy for NAFLD, suggesting that 5f may be worthy of further study.Such prediction of drug-likeness and biological activities before synthesis can decrease the attrition of drug candidates. This highly accessible computational prediction is worthy of popularization and application. Of these novel phenyl ketone derivatives, we identified Drug-likeness values of designed compounds were collected from the SwissADME open-access web tool . Three p1H and 13C NMR spectra. Waters ZQ2000 (USA) was used to determine the MS spectra. FTIR spectra were recorded on a Bruker Tensor II spectrometer (USA). All chemicals were analytically pure and were used without any further purification. The following chemicals were obtained from Energy Chemical (China): 4-butyl-benzoyl chloride, 4-tertbutyl-benzoyl chloride, 1,2,3-trimethoxy-benzene, NBS, and BBr3. The following chemicals were purchased from Fengchuan Chemical (China): DMF, AlCl3, and dichloromethane (DCM). 4-Isobutyl-benzoyl chloride was from TCI Chemicals (China). SOCl2 was from Damao Chemical (China). 1,2-Dimethoxy-benzene was from Aladdin (China).X-4 digital melting point apparatus  was used to record melting points. Bruker Avance (USA) was used to record 2. After dissolution, DMF was added, and the solution was stirred at 80°C for 8 h. After the reaction was stopped, excess SOCl2 was removed by reduced pressure distillation. The resulting solution was washed with DCM.Primarily, 4-butyl-benzoic acid was added to SOCl2a-l was added, followed by AlCl3. After completing the addition, the mixture was brought back to room temperature and stirred for 3.5 hours. The reaction was stopped by adding ice water slowly. The solution was extracted with an equal volume of DCM three times, dried over with Na2SO4, and then dried under vacuum.1,2-Dimethoxy-benzene or 1,2,3-trimethoxy-benzene was added to DCM at 0°C for 10 min. The above-obtained 3a-l was dissolved in DCM. For chloro-substitution, a solution of SO2Cl2 and DCM was added at room temperature. For bromo-substitution, NBS was then added. The reaction was performed for 3 h. The solution was extracted with DCM three times, dried over Na2SO4, and then dried under vacuum.The above obtained 4a-l was dissolved in DCM and stirred for 10 min. After that, BBr3 was slowly added. The reaction was performed at room temperature for 2 h. The reaction was stopped by adding ice water. The solution was extracted with DCM three times, dried over Na2SO4, and then dried under vacuum.The above obtained 1H and 13C NMR, MS, and IR, which were provided in Supplementary Material. The analyses of these spectral data were as follows.All twelve compounds were synthesized and confirmed by 1H NMRδ/ppm : 7.72 , 7.25 , 7.08 , 7.00 , 6.83 , 6.80 , 2.70–2.64 , 1.62 , 1.36 , 0.93 . 13C NMRδ/ppm : 197.91 (CO), 150.45 (Ar C), 147.01 (Ar C), 143.17 (Ar C), 133.67 (Ar C), 131.76 (Ar C), 131.01 (Ar C), 128.79 (Ar C), 119.99 (Ar C), 116.36 (Ar C), 110.13 (Ar C), 35.88 (CH2), 33.11 (CH2), 22.36 (CH2), 13.89 (CH3). MS (M−): 349.22. IR cm−1: 3241 br m, 2360 m, 1649 s, 1601 s, 1278 s, 1175 s, 598 s.Green solid. Yield: 45.5%. M.P.: 88.0-89.2°C. 1H NMRδ/ppm : 12.56 , 7.59 , 7.43 , 7.33 , 6.44 , 6.07 , 2.74–2.68 , 1.66 , 1.40 , 0.96 . 13C NMRδ/ppm : 199.71 (CO), 150.68 (Ar C), 148.02 (Ar C), 146.66 (Ar C), 134.76 (Ar C), 132.38 (Ar C), 129.30 (Ar C), 128.60 (Ar C), 128.03 (Ar C), 114.12 (Ar C), 99.39 (Ar C), 35.71 (CH2), 33.29 (CH2), 22.38 (CH2), 13.93 (CH3). MS (M−): 365.15. IR cm−1: 3206 br m, 1590 s, 1429 s, 1289 s, 1180 s, 887 s.Brown solid. Yield: 47.5%. M.P.: 58.0-59.2°C. 1H NMRδ/ppm : 7.62 , 7.34 , 6.87 , 2.66 , 1.56 , 1.30 , 0.89 . 13C NMRδ/ppm : 193.65 (CO), 148.59 (Ar C), 148.32 (Ar C), 144.27 (Ar C), 134.53 (Ar C), 129.72 (Ar C), 128.62 (Ar C), 128.37 (Ar C), 120.22 (Ar C), 116.54 (Ar C), 116.39 (Ar C), 34.79 (CH2), 32.67 (CH2), 21.72 (CH2), 13.69 (CH3). MS (M−): 303.34. IR cm−1: 3122 br w, 1647 m, 1607 m, 1276 s, 608 m.Brown solid. Yield: 47.2%. M.P.: 54.8-55.2°C. 1H NMRδ/ppm : 12.56 , 7.58 , 7.32 , 7.26 , 2.69 , 1.72–1.57 , 1.38 , 0.95 . 13C NMRδ/ppm : 199.81 (CO), 150.29 (Ar C), 147.98 (Ar C), 145.88 (Ar C), 134.78 (Ar C), 132.63 (Ar C), 129.27 (Ar C), 128.58 (Ar C), 125.03 (Ar C), 113.14 (Ar C), 111.43 (Ar C), 35.70 (CH2), 33.28 (CH2), 22.37 (CH2), 13.92 (CH3). MS (M−): 319.35. IR cm−1: 3178 br m, 1620 s, 1288 s, 1181 s, 647 s.Yellow solid. Yield: 42.1%. M.P.: 115.2-115.9°C. 1H NMRδ/ppm : 9.78 , 7.63 , 7.32 , 6.87 , 6.81 , 2.53 , 1.88 , 0.87 . 13C NMRδ/ppm : 193.68 (CO), 148.35 (Ar C), 147.39 (Ar C), 144.26 (Ar C), 134.61 (Ar C), 129.57 (Ar C), 129.27 (Ar C), 128.35 (Ar C), 120.26 (Ar C), 116.49 ), 44.43 (CH2), 38.94 (CH), 22.08 (CH3). MS (M−): 347.18. IR cm−1: 3239 br m, 1649 s, 1601 s, 1280 s, 1176 s, 756 s.Brown solid. Yield: 46.7%. M.P.: 104.2-105.6°C. 1H NMRδ/ppm : 12.55 , 7.59 , 7.43 , 7.29 , 6.27 , 5.85 , 2.57 , 1.94 , 0.95 , 0.94 . 13C NMRδ/ppm : 199.68 (CO), 150.65 (Ar C), 146.85 (Ar C), 146.48 (Ar C), 134.81 (Ar C), 132.35 (Ar C), 129.28 (Ar C), 129.17 (Ar C), 127.98 (Ar C), 114.14 (Ar C), 99.38 (Ar C), 45.41 (CH2), 30.15 (CH), 22.40 (CH3). MS (M−): 365.26. IR cm−1: 3341 br m, 1607 m, 1287 s, 1178 s, 882 s, 781 s.Brown solid. Yield: 45.7%. M.P.: 137.0-138.0°C. 1H NMRδ/ppm : 7.63 , 7.61 , 7.32 , 7.30 , 6.87 , 6.80 , 2.53 , 1.87 , 0.86 . 13C NMRδ/ppm : 193.68 (CO), 148.35 (Ar C), 147.39 (Ar C), 144.26 (Ar C), 134.61 (Ar C), 129.57 (Ar C), 129.27 (Ar C), 128.35 (Ar C), 120.26 (Ar C), 116.56 (Ar C), 116.42 (Ar C), 44.43 (CH2), 29.47 (CH), 22.09 (CH3). MS (M−): 303.33. IR cm−1: 3242 br m, 1648 s, 1279 s, 758 s.Brown solid. Yield: 50.3%. M.P.: 105.0-105.7°C. 1H NMRδ/ppm : 12.82 , 7.84 , 7.83 , 7.54 , 7.52 , 2.81 , 2.18 , 1.19 . 13C NMRδ/ppm : 199.83 (CO), 150.24 (Ar C), 146.82 (Ar C), 145.82 (Ar C), 134.79 (Ar C), 132.59 (Ar C), 129.25 (Ar C), 129.16 (Ar C), 125.05 (Ar C), 113.14 (Ar C), 111.45 (Ar C), 45.39 (CH2), 30.13 (CH), 22.38 (CH3). MS (M−): 319.35. IR cm−1: 3333 br m, 1606 m, 1288 s, 1182 s, 900 m, 760 m.Yellow solid. Yield: 42.8%. M.P.: 138.5-139.2°C. 1H NMRδ/ppm : 9.95 , 7.65 , 7.56 , 6.87 , 6.79 , 1.31 . 13C NMRδ/ppm : 193.58 (CO), 156.56 (Ar C), 148.32 (Ar C), 144.24 (Ar C), 134.26 (Ar C), 129.58 (Ar C), 128.33 (Ar C), 125.56 (Ar C), 120.24 (Ar C), 116.57 (Ar C), 116.40 (Ar C), 34.88 (C(CH3)3), 30.76 (CH3). MS (M−): 349.13. IR cm−1: 3219 br m, 2920 s, 1649 s, 1599 s, 1280 s, 878 s.Brown solid. Yield: 42.5%. M.P.: 113.0-114.5°C. 1H NMRδ/ppm : 12.58 , 7.62 , 7.54 , 7.45 , 1.38 . 13C NMRδ/ppm : 199.65 (CO), 156.08 (Ar C), 150.66 (Ar C), 146.49 (Ar C), 134.52 (Ar C), 132.35 (Ar C), 129.14 (Ar C), 128.00 (Ar C), 125.57 (Ar C), 114.15 (Ar C), 99.38 (Ar C), 35.16 (C(CH3)3), 31.16 (CH3). MS (M−): 365.31. IR cm−1: 3476 br m, 1603 s, 1265 s, 885 s.Brown solid. Yield: 40.0% M.P.: 131.3-132.5°C. 1H NMRδ/ppm : 7.65 , 7.63 , 7.56 , 7.54 , 6.87 , 6.79 , 1.30 . 13C NMRδ/ppm : 193.58 (CO), 156.56 (Ar C), 148.32 (Ar C), 144.24 (Ar C), 134.26 (Ar C), 129.58 (Ar C), 128.33 (Ar C), 125.56 (Ar C), 120.24 (Ar C), 116.57 (Ar C), 116.40 (Ar C), 34.88 (C(CH3)3), 30.76 (CH3). MS (M−): 303.33. IR cm−1: 3234 br m, 1648 s, 1282 s, 725 s.Brown solid. Yield: 45.8%. M.P.: 116.3-117.1°C. 1H NMRδ/ppm : 12.61 , 7.59–7.52 , 6.57 , 6.24 , 1.40 . 13C NMRδ/ppm : 199.82 (CO), 156.04 (Ar C), 150.29 (Ar C), 145.90 (Ar C), 134.52 (Ar C), 132.61 (Ar C), 129.11 (Ar C), 125.53 (Ar C), 125.08 (Ar C), 113.14 (Ar C), 111.45 (Ar C), 35.15 (C(CH3)3), 31.14 (CH3). MS (M−): 319.34. IR cm−1: 3272 br m, 1605 s, 1283 s, 902 s, 780 s.Yellow solid. Yield: 39.2%. M.P.: 72.8-73.6°C. 2. After growing HepG2 cells overnight, the cells were treated with 500 μM PA  for 24 h. For pharmacological treatments, HepG2 cells were treated with different concentrations of compound 5f simultaneously. The treated cells were subjected to a cell viability and TC/TG content assay. Cell viability was measured by MTT assays. 10 μl MTT  solution was added and incubated for 4 hours. Media were removed and replaced with 100 μl of DMSO. The absorbance was measured at 490 nm using a microplate reader . EC50 values were calculated. To determine TG/TC contents, the cells were gently washed with phosphate-buffered saline and disrupted by sonication. The contents were normalized to total protein using a BCA protein assay kit  and quantified based on the instructions on the corresponding assay kit .HepG2 cells purchased from Procell (China) were grown in DMEM  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin at 37°C and 5% COn = 6) or a high-fat diet  for four weeks. After successfully modeling, positive control silybin (40 mg/kg) or compound 5f  solved in vegetable oil was administrated intragastrically to the assigned HFD groups once every day for three weeks. The dosage for gavage is calculated via the conversion method of human and mice in “Pharmacological Experimental Methodology” edited by Professor Xu Shuyun. Meanwhile, the control and model groups were administered the same amount of vegetable oil (vehicle). Mice were anesthetized with isoflurane inhalation anesthesia. The blood was sampled from the orbital venous. The levels of TG and TC in serum were investigated according to the manufacturer's protocols. Liver samples were fixed in buffered formalin (10%) overnight and embedded in paraffin. 3~5 μm thick serial sections were made from paraffin-embedded tissue and then stained with hematoxylin and eosin (H&E) according to standard protocols.36 male ICR mice  were fed adaptively for one week and then were randomly divided into six groups. Animals were given either a normal chow diet  SDS, 100 mM Tris/HCl, and 1 mM DTT. The samples were quantitated by BCA Assay Kit . Subsequently, the proteins were digested by adding trypsin according to the filter-aided sample preparation described by Matthias Mann. The peptides were then desalted on C18 Cartridges  and resolubilized in 40 μl of 0.1% formic acid. The resulting peptide mixtures were labeled using TMT reagents according to the instructions provided by the company . The LC-MS/MS analysis was conducted on an Easy-nLC Liquid Chromatographer  coupled online to a Q Exactive mass spectrometer  at Shanghai Applied Protein Technology Co., Ltd. LC was performed with a constant flow of 300 nL/min using a C18-reversed phase column  connected to a reversed-phase analytical column . Mobile phase A was water with 0.1% formic acid. Mobile phase B was 84% acetonitrile and 0.1% formic acid. The mass spectrometer was run in positive ion mode and a data-dependent mode. Full scan (m/z 300–1800) was obtained at a resolution of 70,000 at m/z 200, followed by high energy collisional dissociation at a resolution of 17,500 at m/z 200. The automatic gain control values were set to 3e6. Dynamic exclusion duration was 40.0 s. The raw HPLC-MS/MS data were searched using MASCOT 2.2  embedded into Proteome Discoverer 1.4. Maximum missed cleavages were set to 2 and enzyme specificity to trypsin. The FDR values for peptides were 0.01.The model and https://www.pdbus.org/). The PDB IDs for nineteen DEPs are listed in The crystal structure of the proteins was downloaded from Protein Data Bank database .The data were presented as the mean ± standard error (mean ± SD). The difference between groups was analyzed with one-way ANOVA.


Emerging evidence suggests a possible association between hyperglycemia and dolutegravir (DTG), a preferred first-line antiretroviral agent in sub-Saharan Africa (SSA). There is need for rigorous studies to validate this association in the face of increasing DTG use and burden of non-communicable diseases among people living with HIV (PLHIV). We conducted a case–control study to assess the risk of hyperglycemia associated with use of DTG among PLHIV attending Mulago ISS Clinic in Kampala. Cases had hyperglycemia while controls had no hyperglycemia as confirmed by fasting plasma glucose and oral glucose tolerance tests. Demographic, laboratory, and clinical data were collected using interviewer-administered questionnaires and medical record abstraction. Analysis compared cases and controls on DTG use prior to diagnosis of hyperglycemia while controlling for potential confounders using multivariable logistic regression. We included 204 cases and 231 controls. In multivariable analysis, patients with prior DTG use had seven times greater odds of subsequent diagnosis of hyperglycemia compared to those who had non-DTG-based regimens . The odds of hyperglycemia also increased with age  and hypertension . Our study demonstrates a strong association between prior DTG exposure and subsequent diagnosis of hyperglycemia. Given the benefits of DTG, wide-scale use, and the growing burden of diabetes mellitus (DM) in SSA, there is need for systematic screening for hyperglycemia and consideration of alternate regimens for those at risk for DM. It has superior potency for suppressing HIV viral load, a higher barrier to HIV resistance, and better tolerability compared to older ART such as the non-nucleoside reverse transcriptase inhibitors (NNRTIs).13 DTG-based ART regimens are progressively replacing efavirenz(EFV) containing ART regimens, the once preferred first-line ART.11 Due to these benefits, the World Health Organization (WHO) recommended DTG as the preferred first- and second-line ART for all adults, adolescents and children and DTG-based ART has been adopted in many low- and middle-income countries.14 Following WHO’s recommendation, the Uganda Ministry of Health (MoH) in its 2018 HIV treatment guidelines recommended the rollout of DTG-based ART as the preferred first- and second-line ART in the country.15 By the end of 2018, over 17 countries in sub-Saharan Africa had adopted and scaled up DTG-based ART.16To increase the safety and effectiveness of HIV treatment, newer ART medications that are better tolerated have emerged over the past decades.20 A more recent cross-sectional study conducted in Zambia found that when compared to NNRTI’s, DTG-based ART was associated with double the risk of developing metabolic syndrome among PLHIV; metabolic syndrome consists of hyperglycemia, hypertension, and dyslipidemia among other parameters all of which are known to increase the risk of DM.21 In Uganda and throughout SSA, where DTG is being widely rolled out and there is an increasing burden of NCDs among PLHIV and the general population, there is a paucity of information on the association between use of DTG-based ART and hyperglycemia.4 Those who develop hyperglycemia following use of DTG-based ART may need to be considered for alternate ART regimens, such as NNRTI-based which are better tolerated in this regard.15 Because changing the first- and second-line ART recommendations for persons at risk for DM would have profound implications for HIV care in SSA, there is great need for rigorous studies to validate the association between DTG and hyperglycemia. To this aim, we conducted a case–control study among adult PLHIV and receiving care at Mulago Immune Suppression Syndrome (ISS) clinic in Kampala, Uganda to determine the risk of hyperglycemia associated with use of DTG-based ART.Despite its benefits, emerging evidence from case reports, case series, and clinical trials have suggested an association between DTG and disorders of glucose metabolism such as hyperglycemia.The study was conducted at Mulago ISS clinic in Kampala, the largest HIV care center in Uganda. The clinic provides comprehensive HIV services to over 16,500 adults, adolescents, and children. Mulago ISS clinic adopted the use of DTG in March 2018 following the Uganda MoH guidelines for HIV treatment and prevention. Currently more than half of the total patient population is managed with DTG-based ART.We conducted an unmatched case–control study to assess the risk of hyperglycemia associated with use of DTG-based ART among adult PLHIV who were receiving care from Mulago ISS clinic.24 To strengthen the inference of cause before effect, we excluded all cases whose diagnosis of hyperglycemia was made or known prior to March 2018 . True controls were defined as PLHIV aged 18 years and above who were on ART, receiving HIV care from Mulago ISS clinic, and had normal FPG of less than 100 mg/dl or a normal 2-h OGTT of less than 140 mg/dl.24 Pregnant women were excluded from the study because they were initially not eligible for DTG use in early 2018 due to the suspected risk of teratogenicity. Although they were later declared eligible in the Uganda MoH HIV treatment guidelines of 2020, there may persist some bias against its prescription during pregnancy.15 Additionally, recent studies emphasize the need to be cautious when prescribing DTG to pregnant women and this may also contribute to the bias against its prescription during pregnancy.25True cases were defined as PLHIV aged 18 years and above who were on ART, receiving HIV care from Mulago ISS clinic, and had hyperglycemia as confirmed by fasting plasma glucose (FPG) test of ≥ 100 mg/dl or 2-h oral glucose tolerance test (OGTT) of ≥ 140 mg/dl.ART regimens were categorized into DTG-based ART and non-DTG-based ART. DTG-based regimens were all ART regimens that contained DTG in combination with two nucleoside reverse transcriptase inhibitors (NRTIs) such as tenofovir (TDF), abacavir (ABC), or zidovudine (AZT). Non-DTG-based ART were all regimens that consisted of two NRTIs in combination with another anti-retroviral drug from the NNRTIs class such as EFV, nevirapine (NVP), or from the protease inhibitors class such as lopinavir/ritonavir (LPV/r) or atazanavir/ritonavir (ATV/r).To identify cases, we reviewed clinical and laboratory records to find patients with documented evidence of hyperglycemia or use of diabetic medication such as oral hypoglycemics or insulin. Additionally, we identified those who presented at the triage area and consultation rooms for clinicians with signs and symptoms of hyperglycemia. For every case identified, one control was randomly selected from the electronic health record at Mulago ISS clinic, and their clinical records were reviewed to rule out documented evidence of hyperglycemia.All identified cases and controls were approached, and those who consented to participate in the study were subjected to an FPG test and a 2-h oral OGTT to confirm them as true cases or controls. We recruited participants over a period of 2 months (February-April 2021). For efficiency of recruitment and to enable examination of other associations with hyperglycemia among PLHIV, cases and controls were not matched on age, sex, or other clinical conditions. These potential confounding factors were controlled for through multivariable regression analysis.23We instructed consenting participants to fast for at least 8 hours prior to the glucose tests on the following morning. An FPG test and a 2-h OGTT were done by trained personnel at the clinic according to the 2021 American Diabetes Association guidelines.We obtained data on socio-demographic characteristics, lifestyle, and medical history from individual participants using interviewer-administered questionnaires. We also measured and recorded participants’ blood pressure using a validated automatic blood pressure machine, height using a stadiometer, and weight using a balanced scale. We cross-checked information on the medical history, ART regimens, and dates of ART initiation by reviewing participants’ clinical records.The study protocol was reviewed and approved by the Institutional Review Boards of The AIDS Support Organization , the Uganda National Council of Science and Technology , and Yale University (#2000029496). All procedures performed in the study that involved human participants followed the ethical standards of the institutional and/or national research committee. We obtained written informed consent from all participants.p < .05, those found to be confounding in our analysis, and variables determined from literature to be associated with hyperglycemia were entered into the multivariable models to obtain adjusted estimates. Collinear variables were not included in the model. We used the Hosmer and Lemeshow’s goodness of fit and likelihood ratio tests to identify the final model. To assess the robustness of the final model, forward and backward stepwise approaches to model selection were found to converge on the same set of predictor variables. We considered an alpha of 0.05 for the level of significance and 95% confidence interval to test for the association of prior DTG use and subsequent hyperglycemia. All data analyses were conducted using STATA software version 16.Our analysis entailed describing cases and controls, cross-tabulations, and estimation of bivariate and multivariable logistic regression models. We used chi-square tests to determine significant differences between cases and controls across demographic and clinical characteristics as well as on the main exposure variables of past and current DTG use. We tested for DTG’s association with hyperglycemia while adjusting for other potential associations such as age, sex, increasing BMI, and hypertension. Using bivariate logistic regression analysis, we obtained crude odds ratios (ORs) showing the strength of association between the dependent variable (hyperglycemia) and independent variables. We used multivariable logistic regression analysis to control for confounders and determine other factors associated with hyperglycemia among PLHIV. Variables that were significant in bivariate comparisons at Of 473 patients identified and approached, 456 consented to participate in the study including 218 cases and 238 controls. Twenty-one (14 cases and 7 controls) did not complete confirmatory tests for hyperglycemia and were excluded, leaving 435 participants (204 cases and 231 controls) for analysis. Significant differences existed between cases and controls in the variables of education, current BMI, having hypertension as a comorbidity, previous ART regimen, and current ART regimen . Among t27 The mechanism by which DTG causes deranged glucose metabolism is not well understood, although it is hypothesized that DTG causes insulin resistance by chelating magnesium ions which are needed as cofactors for effective action of insulin.28Our findings in a large HIV care center in an African setting corroborate evidence from prior case reports, case series, and randomised controlled trials that have suggested an association between DTG and deranged glucose metabolism.29 Based on unpublished case reports of hyperglycemia among PLHIV from various HIV care clinics in Uganda following initiation of DTG, the Uganda MoH recommended that all patients diagnosed with, or with risk factors for, DM should not be initiated on DTG. Those who develop DM while on DTG are also to be changed to alternate ART regimens.15 Our study findings collaborate this local evidence that these policy changes were justified. The timely change in policy also likely explains why many cases in this study were not continuing to use DTG-based ART; over 40% of the cases had been discontinued from DTG following the diagnosis of hyperglycemia.Our findings indicate that PLHIV who had previously been prescribed DTG-based ART had seven times greater odds of having hyperglycemia compared to those who had been previously prescribed non-DTG ART regimens, even after controlling for potential confounders such as age, BMI, and hypertension as a co-morbidity. Notably, over 70% of the cases in our study had glucose levels that were diagnostic of DM. DM presents a great challenge to Uganda’s health care system, especially in HIV care clinics that remain underprepared to diagnose and manage DM, as is the case in other low-income countries.30 Stakeholders in HIV service delivery should consider implementing the use of more reliable tests for hyperglycemia screening prior to initiation of DTG.The high number of hyperglycemia cases identified in our study indicates that gaps remain in baseline screening of patients for hyperglycemia prior to use of DTG but also the lack of consistent monitoring for those already using DTG. We used an FPG test and a 2-h OGTT to confirm the glycemic states of our study participants including those whose glucose levels qualified them as pre-diabetic. These assays are more reliable than random and fasting blood glucose tests often used in HIV care facilities in Uganda.31 we also found that older PLHIV (particularly above 56 years) and those with hypertension were more likely to be diagnosed with hyperglycemia compared to younger patients and those without hypertension. Although BMI was not significantly associated with the occurrence of hyperglycemia in multivariable regression analysis controlling for other factors such as age and hypertension as a comorbidity; in bivariate analysis, PLHIV who were currently obese, or overweight were more likely to have hyperglycemia compared to those who had normal current BMI. These findings emphasize the need to screen these high-risk groups for hyperglycemia and hypertension vigilantly and consistently prior to use of DTG and to improve access to routine blood glucose monitoring for patients while on DTG.Consistent with prior studies,19 Additionally, the duration of DTG exposure prior to onset of hyperglycemia could not be accurately measured to determine a dose-response effect. We also acknowledge the limitation of incomplete control for confounders both measured and unmeasured as inherent in the case-control design.As with all case–control studies, causal interpretation of our results is limited compared to prospective studies and clinical trials. While we classified exposure as the use of DTG prior to the diagnosis of hyperglycemia , it is not certain if underlying hyperglycemia existed prior to initiating DTG in all cases. It is also possible that DTG may accelerate a person to develop hyperglycemia when they have an underlying disposition, a factor we are unable to conclusively speak to in our case–control study. Due to the retrospective nature of our study, and a lack of reliable medical records, we were limited in establishing whether hyperglycemia resolved after switching from DTG-with or without treatment for hyperglycemia. Additionally, due to this same limitation of retrospective nature of our study, we were unable to establish changes in weight in relation to DTG exposure for cases and controls. However, we acknowledge that weight gain has been associated with use of DTG-based ART and consequently to an increased risk of deranged glucose metabolism and type II DM among PLHIV.Despite the limitations in deducing the precise timing of events, our study noted a sequence of cases having no prior history of hyperglycemia while using non-DTG-based ART, being switched to DTG-based ART following the national roll out of DTG, and then taking them off DTG after finding hyperglycemia. This sequence provides insights on a possible causal association. In the absence of local trials or data from prospective cohort studies, our retrospective approach, coupled with evidence from other settings, provides an efficient mechanism to validate proposed changes to local guidelines in ART regimens.In conclusion, our study demonstrates a strong association between prior DTG exposure and the development of hyperglycemia. Given the great benefits of DTG, the wide-scale roll-out in SSA countries, and the growing burden of DM in these settings, our data weigh in favor of systematic screening for hyperglycemia prior to initiating DTG, routine glucose monitoring, and the consideration of alternate regimens for those at risk for DM.


Animal experiments show that treadmill exercise 24 h after middle cerebral artery occlusion and reperfusion can inhibit neuronal apoptosis, reduce the volume of cerebral infarction on the third (15.04 ± 1.07% vs. 30.67 ± 3.06%) and fifth (8.33 ± 1.53% vs. 30.67 ± 3.06%) days, and promote the recovery of neurological function on the third (7.22 ± 1.478 vs. 8.28 ± 1.018) and fifth (4.44 ± 0.784 vs. 6.00 ± 0.767) days. Mechanistic studies have shown that treadmill exercise increases the density of microvessels, regulates angiogenesis, and promotes the recovery of nerve function by upregulating the expression of vascular endothelial growth factor and laminin. This study shows that rehabilitation exercise 24 h after stroke is conducive to promoting the recovery of patients’ neurological function, and provides a scientific reference for the clinical rehabilitation of stroke patients.Among cerebrovascular diseases, ischemic stroke is a leading cause of mortality and disability. Thrombolytic therapy with tissue plasminogen activator is the first choice for clinical treatment, but its use is limited due to the high requirements of patient characteristics. Therefore, the choice of neurological rehabilitation strategies after stroke is an important prevention and treatment strategy to promote the recovery of neurological function in patients. This study shows that rehabilitation exercise 24 h after stroke can significantly improve the neurological function (6.47 ± 1.589 vs. 3.21 ± 1.069 and 0.76 ± 0.852), exercise ability (15.68 ± 5.95 vs. 162.32 ± 9.286 and 91.18 ± 7.377), daily living ability (23.37 ± 5.196 vs. 66.95 ± 4.707 and 6.55 ± 2.873), and quality of life (114.39 ± 7.772 vs. 168.61 ± 6.323 and 215.95 ± 10.977) of patients after 1 month and 3 months, and its ability to promote rehabilitation is better than that of rehabilitation exercise administered to patients 72 h after stroke ( Stroke has the characteristics of high morbidity, high disability rate, high mortality, and heavy disease burden at a global level. Cerebral ischemic stroke (CIS) accounts for 80% of all stroke patients, is a common type of clinical stroke, and is also the leading cerebrovascular accident leading to patient death and disability . After sStroke is one of the major causes of long-term disability, and effective post-stroke rehabilitation is beneficial to the recovery of neurological function in moderate or severe stroke victims . ConstraEvidence-based early intervention of rehabilitation measures can improve the daily self-care ability of post-stroke patients, effectively reduce the neurological dysfunction of patients, and significantly improve the quality of life, thereby reducing potential medical care costs and saving social resources . AccordiIn this study, stroke patients were selected as the research objects, and two timepoints, 24 h and 72 h after stroke, were selected as the start times for rehabilitation training. The effects of early rehabilitation training at different timepoints on the neurological function of patients with acute ischemic stroke were compared. In addition, this study took the middle cerebral artery occlusion (MCAO) rat model as the research object to explore the molecular mechanism of early rehabilitation training to promote neurological recovery. The purpose of this study is to provide a scientific, targeted, and rational basis for the selection of early rehabilitation training timing for patients with acute ischemic stroke.p < 0.05) (p < 0.05)  . The FMA < 0.05) .p > 0.05). At 1 month and 3 months of rehabilitation, the ADL scores of the patients in the 24 h rehabilitation group were 66.95 ± 4.707 and 96.55 ± 2.873, respectively, which were higher than those in the 72 h rehabilitation group (52.95 ± 6.725 and 84.46 ± 6.971), and the difference was extremely significant (p < 0.001) (p < 0.001)  . The qua< 0.001) .p < 0.01) and fifth  days in the 24 h treadmill exercise group, and the difference was statistically significant. This shows that rehabilitation training at 24 h after MCAO can reduce the volume of cerebral infarction and reduce brain damage caused by cerebral ischemia-reperfusion . This shows that rehabilitation training 24 h after cerebral ischemia-reperfusion can effectively promote the recovery of neurological function in rats  and fifth (4.44 ± 0.784 vs. 6.00 ± 0.767) days, and the difference was statistically significant  in the rat brain on day 1, day 3, and day 5 in the 24 h treadmill exercise group after MCAO was significantly increased when compared to the MCAO group, and the differences were statistical significance . The expStroke is currently a major cause of disability and death for adults in the world . The patSubstantial progress has been made in the research on post-stroke rehabilitation, and targeted motor function recovery can promote the motor function of ischemic stroke patients ,24,25,26p < 0.05). This suggests that rehabilitation training has a positive effect on the recovery of the nervous system function of acute stroke patients.The results of this study showed that after 1 month of rehabilitation training, the NIHSS score of the rehabilitation training group at 24 h post-stroke was 3.21 ± 1.069 points, and the NIHSS score of the 72 h rehabilitation group was 5.05 ± 1.246 points. After 3 months of rehabilitation, the score of the 24 h rehabilitation group was 0.76 ± 0.852 points, and the score of the 72 h rehabilitation group was 2.51 ± 0.989 points. With the intervention and progress of rehabilitation training, the NIHSS scores of both the 24 h rehabilitation group and the 72 h rehabilitation group were significantly lower than those before rehabilitation, the patient’s neurological function was subsequently significantly improved, and the difference was significant . This shows that the patient’s motor function has been effectively improved with the progress of rehabilitation training. There were significant differences in the FMA scores between the two groups at 1 month of rehabilitation training and 3 months of rehabilitation training (p < 0.05). At 1 month and 3 months of rehabilitation training, the FMA scores of the patients in the 24 h rehabilitation group were significantly better than those in the 72 h rehabilitation group, suggesting that the recovery of motor function of the patients in the 24 h rehabilitation group was faster and better than that of the 72 h rehabilitation group. The Fugl-Meyer Assessment (FMA) scale is an important index for assessing motor function, sensation, balance, joint range of motion, and joint pain in stroke patients . In thisIn the rehabilitation process of stroke patients, the improvement of walking ability is the most frequently concerned aspect by stroke patients and their families. The ability to walk independently is often regarded as one of the influencing factors of patients’ willingness to be discharged from the hospital, and it is also a crucial factor for doctors to judge whether stroke patients can be discharged from the hospital. Gumming et al. pointed out that early rehabilitation training can significantly shorten the number of days for stroke patients to recover their ability to walk independently and accelerate the process of patients’ motor function recovery through a clinical randomized controlled trail. Early rehabilitation training is a fast path for patients with acute stroke to restore independent walking ability, which can directly promote the recovery and discharge of patients and return to life and society . Some stActivities of daily living (ADL) refer to the most basic and most common activities that individuals repeat every day in order to maintain their survival and meet their daily needs, that is, a series of activities necessary for an individual in terms of clothing, walking, eating, housing, transportation, maintaining personal hygiene, and conducting independent social activities . The abiThe results of the quality of life survey showed that the quality of life scores of the patients in the 24 h rehabilitation group were higher than those in the 72 h rehabilitation group at 1 month and 3 months of rehabilitation exercise, suggesting that early rehabilitation training had a positive effect on the improvement of patients’ quality of life, and that rehabilitation training 24 h after stroke is more conducive to the improvement of patients’ quality of life. These effects may be related to the fact that early rehabilitation training helps to promote the recovery of neurological and motor functions and enables patients to better adapt to autonomous life, thereby improving the quality of life of patients.The results of animal experiments in this study showed that the treadmill exercise 24 h after middle cerebral artery occlusion and reperfusion reduced the volume of cerebral infarction in rats, inhibited cell apoptosis, decreased neurological function scores, and enhanced neurological function recovery. This study used the rat MCAO model to investigate the mechanism by which rehabilitation exercise within 24 h after stroke promotes neurological function. A large number of studies have shown that aerobic exercise is beneficial to promote the recovery of neurological function after stroke. Treadmill high-intensity interval training for 20 min a day elicited significantly greater acute increases the level of BDNF in the serum of post-stroke patients and corticospinal excitability . VasculaLaminin is a glycoprotein, which is an important structural component in the extracellular matrix, has multiple functions in the central nervous system, and is widely involved in the development of the nervous system . In addip < 0.05). Animal experimental studies have shown that treadmill exercise 24 h after middle cerebral artery occlusion can promote the recovery of neurological function by upregulating the expression of VEGF, increasing the formation of blood vessels, reducing the apoptosis of neurons, reducing the volume of cerebral infarction, and reducing brain tissue damage. To summarize, rehabilitation training begun 24 h after stroke has a better prognosis for patients with acute ischemic stroke, the time required for patients to achieve functional recovery is shorter, and the effect is better, which can shorten the hospitalization time of patients. Patients and their families save medical expenses and improve social and economic benefits. However, this study also has some shortcomings, such as a small sample size and a short study time. In addition, the patient-specific physiology, including the intactness of circle of Willis and leptomeningeal collateral flow, has important guiding significance for evaluating cerebral perfusion and neurorehabilitation in stroke patients [Although clinical guidelines in recent years have pointed out that rehabilitation training should be as soon as possible post-stroke, the optimal time for rehabilitation is still uncertain. This study emphasizes gradual progress in the rehabilitation program. The results suggest that rehabilitation training at 24 h and 72 h after stroke can effectively improve the patient’s neurological and motor functions, and enhance the patient’s ability to live independently, improving their quality of life. The results of this study also suggest that 24 h rehabilitation training after stroke is more effective than 72 h rehabilitation training in the recovery of various functions in patients with acute ischemic stroke . Basic information on the included patients is summarized in The patients who met the inclusion criteria were randomly divided into the 24 h rehabilitation group with 38 cases and the 72 h rehabilitation group with 37 cases using the random number table method . The genIf the muscle strength was less than or equal to grade 1, the patients were instructed to perform supine position training, including correct placement of good limbs, maintaining anti-spasmodic position, position replacement, bridge exercise, etc. Professional rehabilitation specialists intervened to perform passive movement of limbs and joints for patients. The rehabilitation program was as follows: 30 min/time, 2 times/d, 6 d/week.Grade 1 < muscle strength ≤ grade 3, on the basis of the previous stage, the activities range of joint were changed from large to small, and the amount and frequency of activities were gradually increased. Rehabilitation specialists instructed patients to perform exercises such as stretching hands and elbows, simulating face washing, bending and stretching knee and hip joints, and moving ankle and toe joints . Rehabilitation specialists assisted patients in transitional training from supine to sitting position, training of back muscles, abdominal muscles and respiratory muscles, gradually adding shoulder and foot activities, Bobath handshake training, reflex anti-spasm training, sitting, standing balance training, etc.Muscle strength > 3, with the help of rehabilitation specialists, the rehabilitation training gradually increased to bedside standing exercises, standing balance exercises, and the activity time was gradually extended. According to the state of the patient, the training of sit-ups, active joint training, walking training, fine function training, daily living ability training and occupational therapy, etc., were gradually increased. The rehabilitation program was as follows: 60 min/time, 2 times/d, 6 d/week.The recovery effect of the patients was evaluated before rehabilitation, 1 month after rehabilitation, and 3 months after rehabilitation. ① Neurological function: The NIHSS was used to assess the neurological status, with a total of 42 points, with 0 point representing completely normal, higher scores indicate more severe neurological deficits in patients . ② MotorMale adult Sprague–Dawley rats (SD), weighing 230–250 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  Experiments were carried out after a week of acclimatization in a specific pathogen-free laboratory with controlled temperature (20–25 °C) and humidity (50–60%). Both pelleted chow and water were sterilized and freely eaten and drank by rats. The rats were coded uniformly, and the numbers were randomly selected into the sham group, the MCAO group, and the 24 h rehabilitation training group. The samples were taken at the 1 d, 3 d, and 5 d timepoints after rehabilitation training. All experiments were approved by the Committee of Medical Ethics and Welfare for Experimental Animals, Henan University School of Medicine.Rats were fasted 12 h before surgery. The rats were anesthetized with 1% sodium pentobarbital by intraperitoneal anesthesia. During the operation, the rats were kept breathing spontaneously, and a constant temperature heating pad was used to keep the body temperature of the rats constant (37 ± 0.5 °C). After the rats were anesthetized, the neck fur was shaved, and the surgical site was disinfected by 70% ethanol. An incision was made in the middle of the neck with surgical scissors, and the muscle layer was separated under a dissecting microscope to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A specific suture was inserted from the external carotid artery according to Longa’s classic method, which is detailed as follows: Tie a dead knot in the external carotid artery to close the blood vessel, tie a slip knot to fix the suture, use microscissors to cut a V-shaped incision, insert the suture, and fix by the slip knot. Untie the knot at the internal carotid artery, turn the suture so that the uppermost end of the suture is in the same direction as the internal carotid artery and slowly push the suture along the upper left until a slight resistance is felt. When the blood flow curve of the middle cerebral artery is observed to decrease by about 70–80% by laser doppler flowmeter, the advancing of the suture can be stopped, and the slip knot of the fixed suture can be lightly tied at the same time. After 2 h, the suture was removed and the slip knot on the common carotid artery was opened, which was reperfusion. The operation of the sham group was exactly the same as that of the model group except that the suture was inserted without blocking the blood flow of the middle cerebral artery.Before the rehabilitation program, all rats were given adaptive treadmill exercise for 3 days on treadmill platform. The treadmill speed was set at 15 m/min and the time was set at 30 min/d. All three groups of rats were grasped under the same conditions. After MCAO, the model group and the sham group were put back into their original cages without any treatment and nursing measures, and they were allowed to eat and drink freely. The rehabilitation group were started treadmill training at 24 h after MCAO, and the rehabilitation program was determined as the one formed in the adaptive training programs. The rat brains were sampled on days 1, 3, and 5 after treadmill training for further analysis.After the treadmill training, the rats were anesthetized with 1% sodium pentobarbital by intraperitoneally. The brains were harvested after perfusion with 1 x PBS on ice, and the cerebellum and olfactory bulb were removed. The brains were placed in a brain matrix, frozen at −20 °C for 5–10 min, taken out and cut into 5 coronal slices with a thickness of 2 mm. The brain slices were incubated with 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) in 1 x PBS for 15 min at 37 °C in thermocycler protected from light. The slices were fixed in 4% PFA and photographed by a camera. The normal brain tissue was shown in pink and the infarcted area was shown in white. Image J software analyzed and calculated the infarct volume.Calculation formula: infarct volume = (volume of unlesioned hemisphere − infarct volume of lesioned hemisphere)/volume of unlesioned hemisphere × 100% .Modified neurological severity scores (mNSS) is a common tool for assessing the state of rodents neurological function deficits . The mNSRats were anesthetized with 1% sodium pentobarbital by intraperitoneally, and the brains were harvested by transcardial perfusion, lysed with RIPA buffer , and total protein was extracted. The total protein was quantified by BCA kit, and 30 μg of total protein was loaded, the target protein was separated by 12% SDS-PAGE electrophoresis, and the protein was transferred to PVDF membrane. After protein transfer, the membrane was placed in 5% non-fat milk in 1 X TBST for blocking for 60 min, and the primary antibody was incubated at 4 °C overnight . On another day, the membranes was washed 3 times with TBST for 10 min each time, the secondary antibody was incubated for 90 m at room temperature, and the membrane was washed with TBST 3 times for 10 min each time. ECL ultrasensitive luminescent solution was used for chemiluminescence.Rats were anesthetized with 1% sodium pentobarbital by intraperitoneally, and the brains were harvested by transcardial perfusion, fixed in 4% paraformaldehyde, and embedded in paraffin. Coronal sections (8 μm) of brain were cut with a microtome, dewaxed, rehydrated, and antigen retrieval was performed in citrate buffer using a microwave oven. The sections were incubated with anti-laminin antibody  and anti-VEGF antibody  diluted with 5% skim milk, and placed at 4 °C overnight. On the second day, the wet box was taken out, placed at room temperature for 30 min, and washed three times with PBS for 10 min each time. The excess liquid was wiped off and diluted fluorescent secondary antibody (1:500) was added in the dark. Samples were incubated at room temperature for 3 h and washed 3 times with PBS for 10 min each time. Slides were mounted with DAPI glycerol and observed under a fluorescence microscope and images were captured.2 test, and the measurement data were tested by t-test after the homogeneity of variance obeyed normal distribution, and the nonparametric test was performed if the normal distribution is violated in any subgroup. The statistical method for the comparison of scores before and after rehabilitation within the group was based on the results of the sphere test. The volume of cerebral infarction was analyzed by one-way analysis of variance, and p < 0.05 was considered statistically significant.SPSS 21.0 software was used for comprehensive data analysis, and the data were expressed as the mean ± standard deviation (mean ± SD). The enumeration data were tested by χ


Glycogen synthase kinase-3β (GSK3β) inhibitor enhances bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone defects. The present study aimed to investigate the effect of AR-A014418  on the migration and osteogenic differentiation of rat-derived dental pulp stem cells (rDPSCs), and further explore the underlying mechanism.rDPSCs were isolated from rats, and then cultured with different concentrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration, osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8 assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot, and RT-PCR, respectively.Our present study demonstrated that AR of various concentrations  not only promoted the rDPSC proliferation and migration, but also increased calcium deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers  in rDPSCs. It was also found that the administration of AR resulted in an increase in the expression level of p-GSK3β (Ser), β-catenin, p-PI3K, and p-Akt, and a reduction in p-GSK3β (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced cell migration and osteogenic differentiation of rDPSCs induced by AR.Our results provide evidence that AR significantly promotes migration and osteogenic differentiation of rDPSCs by activating β-catenin/PI3K/Akt signaling pathway. In the last decade, mesenchymal stem cells (MSCs) have shown great potential in the field of regenerative medicine due to their capacities of self-renewal and multilineage differentiation.,,In recent years, increasing studies have found that some small molecules show osteogenic function via diverse signaling mechanisms.in vitro, and preliminarily explore the underlying molecular mechanisms.The aim of this study is to investigate the effects of GSK3β inhibitors, AR, on migration and differentiation of rat DPSCs (rDPSCs) into osteoblasts 2 incubator. The third rDPSC passage was subjected to characterization, which included identification of the cell surface markers and multidirectional differentiation of rDPSCs. Every procedure was approved by the Animal Care and Use Committee of Fujian Provincial Hospital.Sprague–Dawley rats  were purchased from Guangdong Medical Laboratory Center , and rDPSCs were isolated from the dental pulp of incisors of rats, as Alksne et al.4 rDPSCs were maintained in cultured media containing 10 mM β-glycerol phosphate, 50 μg/mL l-ascorbic acid, 10 nM dexamethasone, and 2 mM glutamine for 7 or 14 days. During the cultivation, the media were renewed three times every week.For osteogenic differentiation, 5 × 104 rDPSCs were maintained in cultured media containing 5 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone for 14 days. During the cultivation, the media were renewed every other day.For adipogenic differentiation, 5 × 10ARS staining was conducted to confirm osteogenic differentiation and assess calcium-rich deposits in rDPSCs. In brief, after incubated with osteogenic media for 14 days, rDPSCs were fixed with 4% paraformaldehyde (PFA) for 15 min and subsequently washed with PBS. 2% ARS solution was added and incubated for 15 min. Thereafter, the stained rDPSCs were observed under an optical microscope. For quantitative evaluation, ARS was dissolved in 5% perchloric acid by measuring absorbance at 562 nm under a microplate spectrophotometer.As a fat-soluble diazo dye that can stain lipid droplets in cells, Oil Red O is usually utilized to confirm the adipogenic differentiation of MSCs. Briefly, rDPSCs were fixed with 4% PFA, followed by staining with Oil Red O solution in isopropanol for 15 min. Finally, differentiated oil red O-positive rDPSCs were observed and imaged under a microscope.rDPSCs were lysed in RIPA buffer  on ice to extract total protein. The total protein was separated by SDS-PAGE gels and electrophoretically transferred onto PVDF membranes, which were subsequently blocked in skimmed milk (12%) for 2 h. The membranes were incubated overnight at 4 °C with primary antibodies, and subsequently rinsed three times before incubation with the secondary antibody. Finally, protein bands were visualized using ECL Substrate Kit (Abcam), and quantified using Image J software . The information of antibodies used in this study was described in rDPSCs were cultured in media containing different concentrations  of AR  for 1 day, 3 days, 5 days, and 7 days, respectively. At the indicated time points, a CCK-8 reagent  was added to each well. Finally, the absorbance at 450 nm was measured under a microplate reader  after additional 2-h incubation.Briefly, 700 μL cultured media were added to the bottom chamber. Then, 200 μL rDPSCs suspension (serum-free) with different treatments was added to the top chamber with a filter membrane (8 mm pores), followed by 24-h incubation. The cells which migrated to the bottom chamber were fixed and stained. They were then photographed under a microscope.After incubated with osteogenic media for 7 days, the ALP activity of rDPSCs was determined using colorimetric Alkaline Phosphatase Assay Kit (Abcam). In short, rDPSCs were incubated with ALP substrate for 15 min in the dark. Then, the reaction was stopped by 2 M NaOH. The absorbance of the product was detected at 405 nm under a microplate reader.Total RNA was extracted from rDPSCs by using TRIzol reagent . Thereafter, RT-PCR analysis was conducted as described previouslyAll data were expressed as mean ± standard deviation (SD) of three independent experiments. The Student's t-test was used for comparison between two groups. For multiple-group condition, one-way ANOVA was performed with Bonferroni's method. P < 0.05 indicated significant difference.rDPSCs were isolated from incisors of rats, of which surface antigens were characterized using Western blot. Compared with non-DPSCs, CD29, CD44, CD90, and CD105 were obviously up-regulated, while CD34 and CD45 were down-regulated in the isolated rDPSCs A. ARS stUsing the CCK-8 kit, the effect of AR  on rDPSCs proliferation was observed A. On dayThe biological role of AR in osteogenic differentiation of rDPSCs was also explored using ALP assay, and ARS staining, and the expression of osteogenic markers  was determined. The results showed that the ALP activity of rDPSCs in 2.5 μM and 5 μM groups was significantly higher than that in the control group after osteogenic induction for 7 days C. SimilaTo preliminarily explore the potential mechanisms of AR acting in migration and osteogenic differentiation, the effect of AR on the activity of Wnt/β-catenin, PI3K/Akt, ERK1/2, and JNK signaling pathways in rDPSCs was investigated. The results were depicted in To confirm the role of PI3K/Akt signaling in the function of AR in rDPSCs, rDPSCs were pretreated with LY294002 (15 μM), a specific PI3K inhibitor, for 1 h before AR treatment (5 μM). LY294002 significantly weakened the migration ability of AR-treated rDPSCs A. ALP as,DPSCs are an attractive source of MSCs for regenerative medicine due to their relative accessibility and lack of ethical controversy.In an attempt to determine the ideal concentration range of AR on the cell proliferation of rDPSCs, a CCK-8 assay was carried out on rDPSCs after treated with diverse concentrations of AR. In our study, after 7 days of incubation, AR suppressed cell viability of rDPSCs at a concentration of 10 up to 20 μM, while no more than 5 μM promoted the cell viability. Based on the results, a concentration range of 1–5 μM was chosen for the subsequent experiments. Thereafter, our data demonstrated that the migratory ability of hDPSCs was enhanced by 2.5 and 5 μM of AR. Previous studies indicated that GSK3 inhibitors potentially promoted osteogenic differentiation of several MSCs.It is well known that Wnt/β-catenin signaling is involved in tissue generation, regeneration, as well as self-renewal.,,Moreover, our data also indicated that AR could increase the activity of the PI3K/Akt pathway in a dose-dependent manner, but displayed no obvious effect on JNK signaling in rDPSCs. Our study also revealed that the activity of the ERK1/2 in rDPSCs was obviously elevated only when the concentration of AR reached 5 μM. This suggested that a high dose of AR might further induce the activation of ERK1/2 in rDPSCs. Since our results demonstrated that AR promoted migration and osteogenic differentiation of rDPSCs once its concentration reached 2.5 μM, the subsequent experiments merely focused on the role of the PI3K/Akt pathway in the function of AR. Akt is an important member of the protein kinase family, and it is at the center of the signaling pathway, which is a biological signal transduction pathway initiated by PI3K and can be activated by many stresses. Multiple studies implicated the PI3K/Akt signaling pathway in cell proliferation and differentiation.in vitro. Activation of theβ-catenin/PI3K/Akt signaling pathway was involved in the effect of AR on migration and osteogenic differentiation of rDPSCs.In conclusion, the present study indicated that AR, a GSK3β inhibitor, promoted migration and osteogenic differentiation of rDPSCs The authors have no conflicts of interest relevant to this article.