Capture and delivery of tail-anchored proteins to the endoplasmic reticulum

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal transmembrane segment renders TA proteins inherently prone to aggregation and necessitates their posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Here, we review important new insights into the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Interestingly, several alternative routes by which TA proteins can be targeted to the ER have emerged, raising intriguing questions about how selectivity is achieved during TA protein capture. Furthermore, mistargeting of TA proteins is a fundamental cellular problem, and we discuss the recently discovered quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins.

IntroductionThe protein components of biological membranes expand their functionality beyond physical barriers by acting as gateways, allowing intercompartment communication as well as facilitating transport and other membrane-associated processes. Membrane proteins collectively constitute ∼30% of the proteome of most organisms (Krogh et al., 2001), and their biogenesis represents a major challenge for cells. Their hydrophobic transmembrane domains (TMDs), essential for integration into the lipid bilayer and functionality, render such proteins inherently prone to aggregation in the aqueous cytosolic environment. Dedicated targeting strategies for chaperoning such proteins to their target membranes are therefore necessary. Proteins destined for the ER that carry short signal sequences at their N-terminal end and/or internal TMDs are typically recognized cotranslationally by the signal recognition particle (SRP). This arrests translation and induces relocalization of the ribosome nascent chain complex (RNC) to the ER-bound Sec61 translocon, where the newly synthesized protein is channeled directly into the ER lumen and/or membrane (reviewed in Akopian et al., 2013; Rapoport et al., 2017). In both yeast and mammals, a macromolecular ER membrane protein complex (EMC) cooperates with the translocon by assisting the cotranslational folding and biogenesis of polytopic membrane proteins in the ER as well as itself acting as a membrane insertase, mediating the correct topological insertion of the first TMD of specific G-coupled receptors (Bai et al., 2020; Chitwood et al., 2018; Shurtleff et al., 2018). Furthermore, an SRP-independent ER targeting pathway (SND) has recently been revealed in yeast, where proteins containing TMDs in their central regions are captured by Snd1 and directed toward a Sec61 translocon associated with Snd2 and Snd3 (Aviram et al., 2016). This pathway appears to have a broad client spectrum and has been suggested to functionally compensate when other ER targeting pathways are impaired.Tail-anchored (TA) proteins represent a specific class of membrane proteins characterized by a single TMD close to their C-terminus (reviewed in Kutay et al., 1993). The TA protein family contains >50 members in yeast (Beilharz et al., 2003), >500 in plants (Kriechbaumer et al., 2009), and >300 in humans (Kalbfleisch et al., 2007), which populate different membranes (ER, Golgi, and inner nuclear, outer mitochondrial, and peroxisomal membranes). These proteins fulfill diverse membrane-associated functions ranging from regulating intracellular vesicular trafficking (SNARE proteins) to apoptosis, autophagy, lipid biosynthesis, and protein degradation. The topology of TA proteins dictates their posttranslational targeting, as translation termination occurs concurrent with emergence of the TMD from the polypeptide exit tunnel of the ribosome. A major route to the ER for TA proteins is the evolutionarily conserved guided entry of TA proteins (GET) pathway in yeast and the homologous transmembrane recognition complex (TRC) pathway in mammals (Figs. 1 and ​and2;2; Borgese et al., 2019; Schuldiner et al., 2008; Stefanovic and Hegde, 2007). The established view of the GET/TRC pathway (reviewed in Borgese et al., 2019; Chio et al., 2017) involves the posttranslational capture of TA proteins by a pretargeting complex composed of the homodimeric, cytosolic chaperone Sgt2 (yeast)/SGTA (mammals) and the Get4-Get5 heterodimer (yeast)/TRC35-UBL4A-BAG6 complex (mammals; Jonikas et al., 2009; Mariappan et al., 2011; Wang et al., 2010). Interaction of the pretargeting complex with an ATP-bound form of the ATPase Get3 (yeast)/TRC40 (mammals) results in transfer of the TA protein from Sgt2/SGTA to Get3/TRC40 such that the TMD is shielded within a hydrophobic pocket of Get3/TRC40 (Bozkurt et al., 2009; Mateja et al., 2009; Stefanovic and Hegde, 2007; Suloway et al., 2009). ATP hydrolysis triggered by interaction of Get3/TRC40 with the TA protein, coupled with conformational changes in Get3/TRC40 induced by interaction with Get4-Get5/TRC35-UBL4A-BAG6, drives dissociation of TA protein-bound Get3/TRC40 from the pretargeting complex, allowing delivery to the ER-bound GET receptor composed of Get1 and Get2 (yeast)/tryptophan-rich basis protein (WRB) and calcium-modulating cyclophilin ligand (CAML; mammals; Mariappan et al., 2011; Mateja et al., 2015; Stefer et al., 2011). Receptor binding triggers ADP release and conformational rearrangement of Get3/TRC40, allowing TA protein insertion into the membrane and recycling of Get3/TRC40 (reviewed in Mateja and Keenan, 2018). Although much less is currently known about the GET pathway in plants, homologues of the GET pathway components have been identified or are predicted (Xing et al., 2017; Srivastava et al., 2017; Asseck et al., 2021), and two TA SNARE proteins have been shown to be affected by lack of Arabidopsis thaliana (At)GET3 (Xing et al., 2017). A growing body of evidence indicates functional redundancy between different pathways for targeting TA proteins to the ER (Figs. 1 and ​and2;2; Casson et al., 2017). Although some TA proteins of the secretory pathway fulfill essential cellular functions, deletion of GET pathway components is not lethal in yeast or plants, and for many TA proteins, lack of GET pathway components reduces but does not abolish ER targeting. Consistent with this notion, both the EMC insertase (Guna et al., 2018) and the SND pathway (Aviram et al., 2016) have been shown to support the ER targeting of TA proteins.Open in a separate windowFigure 1.TA protein targeting to the ER in yeast. Nascent TA proteins emerging from the ribosome can be captured by alternative ER-targeting machineries. A major route to the ER is via the GET pathway, involving ribosome-associated capture by Sgt2, followed by Get4/Get5-mediated handover to the Get3 ATPase and insertion into the ER membrane by a heterotetrameric GET receptor complex composed of Get1 and Get2.Open in a separate windowFigure 2.TA protein targeting to the ER in mammals. Mammalian TA proteins are predominantly targeted to the ER by the TRC pathway. After capture by SGTA, together with the BAG6 complex (BAG6, UBL4A, and TRC35), the TA protein is passed to the TRC40 chaperone for delivery to the ER-bound receptor complex formed by WRB and CAML. BAG6 has dual functions bridging ER targeting and ubiquitination of TA proteins and can be antagonized by SGTA. Ubiquitinated TA proteins can be deubiquitinated by ER-associated UPS20/UPS33.Despite a wealth of knowledge on some aspects of TA protein targeting to the ER by the GET/TRC pathway, other features and mechanistic details remain enigmatic. How TA proteins can be captured posttranslationally but also reliably avoid aggregation during handover from the ribosome to the pretargeting complex or other chaperones is an inherent conundrum of their biogenesis. Knowledge on the architecture of the GET/TRC receptor complex and mechanistic understanding of how the TA protein, delivered to the receptor by Get3/TRC40, is inserted into the ER membrane, have been limited by the technically challenging nature of structural analyses of membrane-bound complexes. Furthermore, how the fidelity of TA protein targeting to different membranes is ensured is poorly understood, and little is currently known about how the actions of TA protein targeting and quality control are coordinated. In this review, we describe recent advances that address these key aspects of the GET/TRC pathway and TA protein biogenesis.Capture of nascent TA proteinsIn contrast to cotranslational protein targeting, where capture of client proteins and their delivery to the ER-bound receptor complex is performed by the SRP complex, posttranslational targeting of TA proteins to the ER by the GET/TRC pathway is a more stepwise process, involving a dynamically assembling pretargeting complex that mediates initial capture but then hands over substrates to another chaperone for delivery to the membrane-bound receptor. The existence of a modular pretargeting complex appears to be a feature of the GET/TRC pathway conserved throughout eukaryotes, but compositional and structural differences between species are apparent. In yeast, Get4-Get5 form an obligate heterodimer, whereas the homologous TRC35 and UBL4A interact via an additional component BAG6 (Chang et al., 2010; Chartron et al., 2010; Mariappan et al., 2010; Mock et al., 2015). Biochemical evidence shows that a Get4/TRC35 homologue exists in plants, and homologues of Get5/UBL4A and Sgt2/SGTA are predicted from in silico analyses (Srivastava et al., 2017; Xing et al., 2017). A BAG6 homologue has also been identified in plants (https://www.arabidopsis.org/servlets/TairObject?id=35038&type=locus); however, it remains unknown if this protein associates with components of the GET pathway and/or contributes to TA protein targeting. These differences likely reflect subtle variations in the mechanism of TA protein capture between species and/or the greater need for regulation and surveillance in multicellular organisms.Ribosome binding of pretargeting complex componentsDue to the position of the TMD at the C-terminus of TA proteins, the GET/TRC pathway must capture clients posttranslationally. However, posttranslational capture has the inherent caveat of protein aggregation in the narrow window between emergence of the TMD from the ribosome exit tunnel and protein capture by a chaperoning factor. The first hint how this issue may be overcome came with the intriguing discovery of Get5 in a high-throughput screen for ribosome-associated proteins in yeast (Fleischer et al., 2006). This observation raised the possibility of a physical connection between the upstream components of the GET pathway and the translation machinery, despite the posttranslational nature of the final capture event. Detection of the Get4-Get5 heterodimer associated with polysomes importantly confirmed recruitment of these proteins to actively translating ribosomes (Zhang et al., 2016), further supporting that the GET pathway pretargeting complex might be poised on the ribosomes ready to shield nascent TA proteins directly as they emerge from the exit tunnel. In vitro reconstitution confirmed a high-affinity interaction between Get4-Get5 and ribosomes, and protein–protein and protein–RNA cross-linking analyses pinpointed the polypeptide exit tunnel of the ribosome as the Get4-Get5 binding site (Fig. 1; Zhang et al., 2021). Get4-Get5 bridge interactions between Sgt2 and Get3 to facilitate handover of the TA protein to the downstream chaperone, and therefore functional significance of Get4-Get5 ribosome binding must be coupled to TA protein capture by Sgt2. Indeed, it was recently shown that Get4-Get5 act as a binding platform for recruitment of Sgt2 to ribosomes and that the presence of Get4-Get5 on ribosomes enhances TA protein capture by Sgt2 (Zhang et al., 2021). Crystal structures of GET pathway subcomplexes demonstrate that the N-terminal domains of the Sgt2 homodimer interact with the central UBL domain of Get5, while the N-terminal region of Get5 mediates interaction with C-terminal region of Get4 (Chang et al., 2010; Chartron et al., 2012a; Simon et al., 2013). As Get5 appears to simultaneously interact with Get4, Sgt2, and ribosomes, it is tempting to speculate that ribosome binding of the pretargeting complex may be mediated by the C-terminal region of Get5. However, due to the multimeric nature of the pretargeting complex and evidence that the Get5 C-terminal region also mediates homodimerization (Chartron et al., 2012b), structural analyses of Get4-Get5-Sgt2–bound ribosomes will be necessary to resolve in detail the architecture of GET pathway pretargeting complex–bound ribosomes.The mammalian pretargeting complex components UBL4A, TRC35, BAG6, and SGTA also associate with RNC complexes (Fig. 2; Leznicki and High, 2020; Mariappan et al., 2010), implying that the mechanism of ribosome-associated capture of TA proteins is also employed in mammalian cells to circumvent protein aggregation upon the TMD encountering the aqueous cytosol during targeting. However, the mammalian-specific pretargeting complex component BAG6, rather than the Get5-homologous UBL4A, appears to act as the key ribosome adaptor, and SGTA can also interact with RNC complexes independently of TRC35-UBL4A (Leznicki and High, 2020; Mariappan et al., 2010).Notably, Get3/TRC40, which receive the TA protein from the pretargeting complex, are not ribosome associated (Mariappan et al., 2010; Zhang et al., 2016). This implies that following Get4-Get5–facilitated capture of the TA protein by ribosome-associated Sgt2, the pretargeting complex should dissociate from the ribosome to encounter Get3. It is possible that a conformational change in the pretargeting complex, induced by TA protein binding, triggers release from the ribosome.Recognition of ribosomes synthesizing TA proteinsThe discovery of ribosome-associated populations of the GET pretargeting complex and the BAG6 complex and SGTA in yeast and mammals, respectively, raises the question of how these complexes, which are significantly less abundant than cytosolic ribosomes, are able to identify ribosomes synthesizing TA proteins. Analogous to SRP, which probes the translating ribosome population, preferentially binding ribosomes translating proteins with a signal sequence (Berndt et al., 2009; Holtkamp et al., 2012; Ogg and Walter, 1995; Voorhees and Hegde, 2015), yeast Get4-Get5 show increased association with RNCs containing a TA/TMD in the exit tunnel (Zhang et al., 2021). Mammalian SGTA is likewise selectively recruited to ribosomes synthesizing membrane proteins (Leznicki and High, 2020). This implies that a similar substrate-scanning mechanism, involving transient association events followed by high-affinity docking only onto appropriate ribosomes, is employed in both the co- and posttargeting pathways. The underlying mechanism of how the GET/TRC pretargeting components sense the presence of the TA/TMD in the exit tunnel still remains to be elucidated. Intriguingly, the newly identified ribosome-binding site of the GET pathway pretargeting complex overlaps with that of SRP, implying mutually exclusive ribosome occupancy. Consistent with this, the presence of Get4-Get5 on RNCs with a TMD in the exit tunnel reduces the amount of SRP bound, and strong binding of SRP to an exposed TMD leads to displacement of Get4-Get5 (Zhang et al., 2021). This suggests a compelling model where the GET/TRC pretargeting complex, SRP, and potentially ribosome-bound Snd1 (Fleischer et al., 2006) constantly screen the translating pool of ribosomes and outcompete each other for ribosome binding upon encountering one synthesizing an optimal substrate.Additional players in initial TA protein captureIt is well established both in vitro and in vivo that TA protein targeting by the GET/TRC pathways involves a chaperone exchange in which the TMD, initially shielded by a hydrophobic patch within the C-terminal region of Sgt2, is handed over to Get3, where it is protected within a dedicated hydrophobic groove formed in the ATP-bound state. While Sgt2 has long been considered the upstream component of the GET pathway, it has recently emerged that the abundant, Hsp70-like chaperone Ssa1 can also act as a highly efficient nascent TA protein chaperone and that its effectiveness in TA protein trapping is enhanced by the J domain–containing cochaperone protein Ydj1 (Fig. 1; Cho et al., 2021; Cho and Shan, 2018). Ssa1 and numerous other chaperones physically interact with Sgt2 via its tetratricopeptide repeat domain (Cho and Shan, 2018; Krysztofinska et al., 2017). As this domain of Sgt2 is important for ER targeting of TA proteins in vivo, this supports a potential role of other chaperones alongside Sgt2. Transfer of TA protein cargoes from Ssa1 to Sgt2 is energetically favorable and stimulated by the J domain proteins Ydj1 and Sis1. Interestingly, Ydj1 and Sis1 appear to function redundantly in targeting of a reporter TA protein to the ER (Cho et al., 2021), perhaps suggesting that the chaperone cascade protecting TA proteins during their biogenesis can be more extensive than initially anticipated. However, it still remains to be determined how much of a contribution these proteins make to endogenous TA protein targeting within the native cellular environment. Interestingly, human cells lacking SGTA or BAG6 are viable, and TA protein targeting to the ER can still be accomplished in the absence of these factors (Culver and Mariappan, 2021). It is possible, therefore, for capture by the pretargeting complex to be bypassed, and perhaps in this context, other chaperones, analogous to those characterized in yeast, contribute to the initial protection of nascent TA proteins before their association with TRC40. Along this line, TA proteins with less hydrophobic TMDs, which use the EMC rather than the TRC pathway, have been shown to be chaperoned through the cytosol by calmodulin (Fig. 2; Guna et al., 2018). The existence of alternative, partially redundant, targeting pathways, supported by the nonlethality of yeast, plant, and human GET pretargeting factor knockouts (Stefanovic and Hegde, 2007; Schuldiner et al., 2008; Jonikas et al., 2009; Srivastava et al., 2017; Xing et al., 2017; Culver and Mariappan, 2021), likely helps ensure high-fidelity and robust targeting of TA proteins in vivo.Selectivity of TA protein captureIt has become increasingly clear that nascent TA proteins emerge into a crowded environment where encounters with different machineries can direct them toward different fates, such as targeting to the ER via different routes, targeting to different organelles, or potentially, degradation. These observations suggest a finely tuned process of nascent TA protein capture to direct different proteins to the appropriate destinations and highlight the question of how TA proteins are selectively captured. While all TA proteins share the common characteristic of a single TMD close to the C-terminus that can serve as a targeting signal, the length and hydrophobicity of this TMD, as well as the net charge of the downstream sequence (also termed C-terminal element [CTE]), vary significantly, and these features are important determinants of the ultimate destination of the protein (Beilharz et al., 2003; Borgese et al., 2019). TA proteins of the outer mitochondrial membrane (OMM) and peroxisomes are typified by short, less hydrophobic TMDs, and positively charged CTEs are characteristic features of peroxisomal TA proteins (Horie et al., 2002). In contrast, ER and Golgi TA proteins generally have relatively long, hydrophobic TMDs, and the charge of their CTEs varies considerably (Rao et al., 2016; Borgese et al., 2019). The physiochemical properties of ER TMDs favor capture by Sgt2/SGTA and binding by Get3/TRC40, whereas TA proteins with less hydrophobic TMDs are poor substrates (Coy-Vergara et al., 2019; Guna et al., 2018). This implies that an important layer of capture selectivity is already encoded within the proteins themselves. Strict categorization of different organellar TA proteins based on physiochemical properties is not possible, however, as differently localized TA proteins have partially overlapping properties, indicating that this criterion alone is insufficient to ensure correct targeting.TA proteins not only need to be directed to different target membranes, but they also need to be recognized as bona fide membrane proteins. The hydrophobic sequences that are integral features of membrane proteins must be distinguished from exposed hydrophobic patches of misfolded, nonmembrane proteins that serve as signals for recruitment of the protein quality control machinery. Interestingly, the BAG6 component of the mammalian pretargeting complex sits at the nexus between the alternative fates of targeting and degradation; a minimal C-terminal BAG domain in BAG6 scaffolds interactions between TRC35 and UBL4A and is sufficient for TA protein targeting to the ER (Mock et al., 2015), while the N-terminal UBL domain of the protein promotes recruitment of the ubiquitination machinery to mediate quality control of aberrant proteins (Fig. 2; Rodrigo-Brenni et al., 2014). In this context, BAG6 has been implicated in rerouting SGTA-bound TA proteins that are not efficiently relayed to TRC40 toward the degradation pathway (Shao et al., 2017). SGTA has emerged as another central player in determining the fate of TA proteins, as it is able to antagonize BAG6-facilitated protein ubiquitination. Interestingly, SGTA not only reduces the likelihood of protein ubiquitination by shielding the hydrophobic TMD but also promotes active deubiquitination of BAG6 complex–associated proteins (Leznicki and High, 2012). In this way, SGTA could contribute to the recovery of nascent TA proteins aberrantly marked for degradation by BAG6-associated ubiquitin ligases. Notably, the interplay between the BAG complex and SGTA in determining protein fate extends beyond TA proteins to other membrane proteins targeted cotranslationally (Leznicki and High, 2020). Intriguingly, it was recently shown that ubiquitination of TA proteins can occur independently of BAG6, and that ubiquitinated TA proteins can still be handled by TRC40 and directed to the ER, where they are fully deubiquitinated by USP20/USP33 before or after membrane insertion (Fig. 2; Culver and Mariappan, 2021). It remains unclear mechanistically how exactly ubiquitinated TA proteins evade proteosome-mediated degradation in the cytosol, but it is possible that either the nature of the ubiquitination and/or rapid capture by TRC40 enables them to efficiently reach their destination and be deubiquitinated. It will be interesting to determine if this ubiquitination-deubiquitination cycle simply represents a futile mislabeling and recovery process or whether it fulfils a specific function during TA protein biogenesis.Delivery of TA proteins into the ERBoth the GET/TRC receptor and the EMC complex have emerged as gateways into the ER for TA proteins (Guna et al., 2018; Schuldiner et al., 2008; Vilardi et al., 2011; Yamamoto and Sakisaka, 2012). TA proteins destined for ER insertion via the GET/TRC receptor converge on the homodimeric cytosolic chaperone Get3/TRC40, which escorts them to the ER-bound GET/TRC insertase (McDowell et al., 2020; Wang et al., 2014). Docking of ADP and TA protein–bound Get3/TRC40 onto the GET receptor allows transfer of the TA protein to the receptor, which subsequently mediates their insertion into the membrane (Wang et al., 2014). Then, upon ADP release, Get3/TRC40 is recycled for another round of ATP binding and TA protein targeting (reviewed in detail in Chio et al., 2017).Evolutionary conservation of the GET receptor complexThe Get1 component of the GET receptor is a member of the Oxa1 superfamily of insertase proteins, which includes bacterial YidC and eukaryotic EMC3 (Anghel et al., 2017; McDowell et al., 2021), and it shares its three-TMD topology with other members of this family. Get1 sequence conservation among different phyla readily facilitated the identification of homologues in mammals (WRB) and plants (AtGet1; Srivastava et al., 2017; Xing et al., 2017). In contrast, Get2 homologues are more divergent, and sequence conservation is limited to the functionally essential N-terminal Get3-binding sequence and, to a lesser extent, the three TMDs (Borgese, 2020). CAML has nevertheless been identified as a functional and structural homologue of Get2 in mammals (Yamamoto and Sakisaka, 2012), and demonstrated to complement phenotypes associated with loss of Get1/Get2 when coexpressed with WRB in budding yeast (Vilardi et al., 2014). The existence of Get2 orthologues or functional homologues in other phyla were uncertain for a long time. The recent identification of the archaeplastidic Get2 homologue AtGet2(Asseck et al., 2021) and in silico prediction of Get2 homologues in mollusks and arthropods (Borgese, 2020), however, now strongly support that not only Get1/WRB, but the GET receptor complex as a whole is conserved among eukaryotes.Architecture and stoichiometry of the GET receptorThe interaction between Get1/WRB and Get2/CAML is mediated by their TMDs, which are also necessary for the insertase function of the complex (Vilardi et al., 2014; Wang et al., 2014). Moreover, mammalian WRB and CAML, and likely other homologues as well, are thought to exist as an obligate complex, mutually stabilizing each other. Indeed, the expression levels of WRB or CAML decrease in the absence of the other subunit, likely because of destabilization of the remaining subunit (Colombo et al., 2016; Rivera-Monroy et al., 2016). However, the effects of the individual components seem to be asymmetric on each other. Namely, WRB can insert into the ER membrane correctly in the absence of CAML and is later degraded as an orphan subunit, whereas WRB is required for CAML to assume a correct topology in the first place (Carvalho et al., 2019; Inglis et al., 2020). More specifically, the second TMD of CAML remains exposed to the ER lumen in the absence of WRB, where it acts as a degron, triggering protein degradation. Interestingly, its topology can be corrected posttranslationally, and its degradation prevented when WRB is available in the membrane (Inglis et al., 2020). It remains to be seen whether a similar inter-subunit interplay also occurs in other species; however, results from A. thaliana imply that correct assembly of the GET receptor may be differently controlled in plants, as ectopically expressed AtGet2 remains stable in the absence of AtGet1 (Asseck et al., 2021).Although formation of a Get1/Get2 heterodimer is recognized as a prerequisite for a minimal functional receptor complex, a higher-order stoichiometry of the insertion-competent GET receptor complex has long been actively discussed. Due to the symmetric, homodimeric nature of Get3 in the TA protein–loaded complex, two analogous binding sites for both Get1 and Get2 are offered (Stefer et al., 2011). Despite a partial overlap of the Get1 and Get2 interaction sites on Get3, simultaneous binding of Get3 by Get1 and Get2 has been observed in crystal structures of Get3, together with the cytosolic domains of Get1 and Get2 (Stefer et al., 2011). This gave rise to the notion of a heterotetrameric structure of the GET receptor composed of two Get1 and two Get2 subunits, which could bind a single Get3 dimer with high affinity (Mariappan et al., 2011; Stefer et al., 2011). However, results obtained with in vitro reconstituted proteoliposomes demonstrated that a single dimer of Get1/Get2 can be sufficient for insertion of TA substrates into the membrane (Zalisko et al., 2017). Nevertheless, the heterotetrameric arrangement is supported by recent high-resolution cryo-EM structures of Get3/TRC40 homodimers docked onto the yeast and mammalian GET receptors, which reveal the formation of a heterotetrameric receptor complex upon Get3/TRC40 binding (Figs. 1, ​,2,2, and ​and3;3; McDowell et al., 2020). These new structures further consolidate the previously proposed model (Mariappan et al., 2011; Stefer et al., 2011) that Get3/TRC40 is initially captured by the extended cytosolic domains of Get2/CAML before contacting Get1/WRB (Figs. 1, ​,2,2, and ​and3).3). This arrangement, coupled with the finding that Get1-Get2 can simultaneously bind two Get3 molecules (McDowell et al., 2020), opens the possibility for a relay system where translocation of a first Get3/TRC40-TA protein complex to Get1 immediately allows capture of a second substrate complex by Get2, potentially increasing the efficiency of receptor complex loading and minimizing the risk of mistargeting. Interestingly, the GET/TRC receptor seems to be stabilized at the heterotetramer interface by not only protein–protein but also protein–lipid interactions, indicating that the lipid environment of the ER membrane may also influence the oligomeric state of the receptor. Importantly, complementation experiments in yeast demonstrate that disrupting lipid binding and thus the formation of the heterotetramer leads to in vivo loss of function of the receptor manifesting in TA protein mislocalization (McDowell et al., 2020). Therefore, although a Get1/Get2 dimer appears to be sufficient for the insertase function of the receptor in vitro, it is highly likely that a tetrameric complex is required to ensure efficient and accurate TA protein targeting within the cellular environment.Open in a separate windowFigure 3.Architecture of the Get1/WRB and Get2/CAML in the GET/TRC receptor complex. Get1/WRB and Get2/CAML both possess three TMDs (labeled 1–3) and rely on each other for stability and correct assembly within the ER membrane. The cytosolic regions of Get2/CAML (N-terminus and a loop between TMD 2 and 3) and a cytosolic region of Get1/WRB between TMDs 1 and 2 are docking sites for Get3/TRC40 carrying a TA protein cargo. A hydrophilic groove formed by the Get1/WRB TMDs and Get2/CAML TMD3 is proposed to serve as an insertion route for TA proteins to enter the membrane. Assembly of the heterodimer shown here into the final heterotetrameric structure of the receptor upon Get3/TRC40 binding involves protein–lipid interactions.Mechanistic view of TA protein insertion into the lipid bilayerRecent structural advances provide exciting mechanistic insights into the details of TA insertion, both by the GET/TRC receptor (McDowell et al., 2020) and the EMC complex (Bai et al., 2020; Miller-Vedam et al., 2020; O’Donnell et al., 2020; Pleiner et al., 2020). In the case of the GET/TRC receptor, within the membrane, the TMDs of WRB, together with TMD3 of CAML are arranged such that a hydrophilic groove, sealed at the luminal face but accessible from the cytosol, is assembled (Fig. 3). This likely serves as a substrate entry point with the charged, extreme C-terminus of the TA protein drawn in by interactions with the numerous hydrophilic residues of the receptor channel, thus bringing the TMD in close proximity to the destabilized bilayer, allowing insertion. This corroborates previous results obtained by cross-linking nascent TA substrates to the receptor in vitro, which pinpointed the region around the hydrophilic groove as the entry point for TA proteins into the membrane (Wang et al., 2014). Interestingly, assembly of the GET receptor as a heterotetrameric complex means that tandem hydrophilic grooves generated by each Get1/WRB-Get2/CAML pair represent two alternative routes into the membrane. The asymmetric binding of the TA protein within the Get3 dimer (Mateja et al., 2015), means that depending on the orientation of the docking, insertion via a particular channel will be favored. Dynamic modeling of interactions between the receptor and the TRC40 dimer in different conformations/nucleotide-bound states (Mateja et al., 2015; Stefer et al., 2011) suggest that transition of Get3/TRC40 to the open conformation leads to rearrangement of complex such that the C-terminus of the released TA protein is oriented toward the hydrophilic groove (McDowell et al., 2020). Complementary structural analyses of the EMC complexes (Bai et al., 2020; Miller-Vedam et al., 2020; O’Donnell et al., 2020; Pleiner et al., 2020) reveal analogous hydrophilic groove features, indicating that a common insertion mechanism is used by evolutionarily distant membrane receptors to accomplish insertion of a diverse set of membrane proteins (Bai and Li, 2021; McDowell et al., 2021). For the GET/TRC receptor, it is not yet clear how exactly the TA protein transits from this hydrophilic groove to become fully immersed in the membrane, but an amphipathic helix of Get1/WRB that lies close to the membrane has been suggested to cause membrane distortions that could facilitate TA protein integration.Another key feature of the GET receptor revealed by the new structures is a short helix, α3′, present in the cytosolic domains of both yeast Get2 and human CAML, which binds the TA-binding domain of Get3/TRC40 and serves a gating function for the hydrophilic groove (Fig. 3; McDowell et al., 2020). Contact between the Get2/CAML helix and the Get3/TRC40 TA-binding domain induce conformational rearrangements crucial to the release and insertion of TA substrates in vivo, further underlining the role the GET receptor plays in stimulating substrate release from Get3. It remains to be determined whether, mechanistically, this helix facilitates TA protein insertion by preventing backsliding of the TMD out of the hydrophilic groove or actively driving the TA protein into the channel.Quality control and rescue of TA protein targetingThe multiple possible destinations for TA proteins, together with the broad spectrum of physical properties of secretory pathway TA proteins, the existence of alternative ER targeting and insertion strategies, and the complexity of ensuring optimal capture, renders targeting of TA proteins to the ER inherently prone to errors. Defects in this process can manifest as either redirection of ER TA proteins to other membranes or the spurious ER insertion of non-ER TA proteins. Given the importance of TA protein functions and the toxic effects of cytosolic protein aggregates, either of these scenarios can have seriously detrimental effects on cells, necessitating robust surveillance, recovery, and degradation pathways (Jiang, 2021).Removal of TA proteins misdirected to the OMMIn contrast to ER TA proteins, some TA proteins of the OMM have been proposed to be inserted directly, potentially due to the specific lipid composition of the OMM (Figueiredo Costa et al., 2018). The hydrophobicity of their TMDs are lower than that of secretory pathway TA proteins (Borgese et al., 2001; Beilharz et al., 2003), which, considering that high TMD hydrophobicity is necessary for recognition by the GET pathway (Guna et al., 2018), helps explain how they avoid capture and subsequent delivery to the ER by Get3. In contrast, peroxisomal TA proteins can either use the Pex19-Pex3 machinery to directly reach peroxisomes (reviewed in Mayerhofer, 2016) or be first targeted to the ER by the GET pathway (Schuldiner et al., 2008) before reaching peroxisomes (Fig. 4).Open in a separate windowFigure 4.Quality control machineries regulating distribution of mislocalized TA proteins between the ER and other organelles. ER-destined and peroxisome (Perox.)-destined TA proteins can mislocalize to the OMM, and OMM TA proteins can mislocalize to the ER. ATP-dependent machines in these membranes can recognize and displace mistargeted protein while correctly localized proteins are retained, for example by interactions with binding partners (indicated by unnamed, colored circles on the OMM). Nomenclature is as in yeast.It has been observed that ER-inserted TA proteins can mislocalize to mitochondria when the functionality of the GET pathway is impaired (Schuldiner et al., 2008). A salient example is yeast Pex15, which is a peroxisome-destined TA protein first inserted into the ER but that mislocalizes to mitochondria not only when the C-terminal 30 amino acids or Pex19 are lacking, but also in the absence of Get3 (Schuldiner et al., 2008; Okreglak and Walter, 2014; Li et al., 2019). Furthermore, a basal level of mistargeting of secretory pathway TA proteins, such as yeast Gos1, to the OMM is observed even in the presence of a functional GET/TRC pathway, implying that some level of mistargeting is unavoidable (Chen et al., 2014). This therefore raises the question of how mistargeted TA proteins are recognized and cleared while the appropriately localized proteins are retained.A mechanism by which TA proteins incorrectly inserted into the OMM can be extracted has recently been described; the highly evolutionarily conserved mitochondrial and peroxisomal AAA-ATPase Msp1 (ATAD1 in mammals) has been shown to be essential for the removal of mitochondrially mislocalized Pex15 and is thought to act as a general dislocase of TA proteins mislocalized to the mitochondria (Fig. 4; Chen et al., 2014; Okreglak and Walter, 2014). This explains why double-mutant yeast strains lacking both GET pathway components and Msp1 mislocalize TA proteins noticeably to mitochondria (Li et al., 2019). Accordingly, GET pathway components and Msp1 show a synthetic negative genetic effect (Chen et al., 2014; Okreglak and Walter, 2014), emphasizing the importance of high-fidelity protein targeting and quality control. Msp1 forms hexamers in the OMM, and in vitro, its ATPase activity is sufficient to drive the removal of TA proteins from proteoliposomes (Wohlever et al., 2017), demonstrating that Msp1 alone can both recognize its substrates and drive their subsequent extraction from the membrane. Recent visualizations of Msp1-substrate complexes provide insights into the mechanism of membrane extraction; the TA protein substrate enters via a single, hydrophobic site and is then anchored within the hydrophobic pore by a network of aromatic amino acids (Wang et al., 2020). ATP hydrolysis, the driving force for membrane extraction, is coordinated with subunit positioning within the complex via specific elements at the subunit interfaces (Wang et al., 2020). Interestingly, based on in vitro experiments with MBP-tagged Pex3, it has been suggested that the unfoldase activity of Msp1 may be regulated by Pex3 on peroxisomes (Castanzo et al., 2020), but it remains to be determined whether a similar regulatory mechanism exists in the OMM as well.The stringency of recognition of mitochondrially mislocalized TA proteins relies on at least a twofold recognition mechanism by Msp1. First, basic residues typical of the luminal tails of peroxisomal TAs are recognized when exposed in the mitochondrial intermembrane space (Li et al., 2019). Second, exposed hydrophobic amino acids close to the membrane, present in various secretory pathway and peroxisomal TA proteins, also act as a recognition signal for Msp1 (Li et al., 2019). Furthermore, there is evidence indicating that orphan TA proteins lacking binding partners within the OMM are more readily recognized and displaced by Msp1 (Weir et al., 2017; Dederer et al., 2019). This indicates that besides the biophysical properties of Msp1 substrates, their ability to form functional protein–protein interactions with other membrane proteins is an important discriminating factor that determines whether a TA protein is retained in or removed from the OMM. Mechanistically, lack of a binding partner may render mislocalized TA proteins less stably anchored within the membrane, or, when separated from their normal interaction partners, mistargeted proteins may be more likely to expose hydrophobic patches to the cytosol, both of which would increase the chance of expulsion by Msp1.It is possible that after removal from the OMM by Msp1, secretory pathway TA proteins returned to the cytosol could be retargeted if they are captured by Get3/TRC40 or other chaperones capable of directing them to the ER-bound insertase machineries. However, a cellular machinery also needs to exist that degrades excess TA proteins in the cytosol or at the ER to ensure protein homeostasis. Indeed, the E3 ubiquitin ligase Doa10, an important player in ER-associated degradation, has recently emerged as a quality control factor responsible for sensing and ubiquitination of spurious and excess TA proteins ejected from the OMM. It has been suggested that Doa10-mediated ubiquitination may take place either in the cytosol (Dederer et al., 2019) or after retargeting to the ER, where their extraction and degradation is facilitated by the AAA-ATPase Cdc48 (Matsumoto et al., 2019; Fig. 4).Mistargeting of mitochondrial TA proteins to the ERIt is not only the case that nonmitochondrial TA protein are misdirected to the OMM; also, reciprocal events can occur as OMM TMD-containing proteins are observed in the ER, especially when mitochondrial targeting signals are masked, when the mitochondrial import machineries are overloaded, or upon mitochondrial dysfunction (Hansen et al., 2018; Xiao et al., 2021; Vitali et al., 2018). More specifically, the GET pathway itself has been shown to contribute to the ER mislocalization of OMM TA proteins (Vitali et al., 2018; Xiao et al., 2021). While the high efficiency of mitochondrial targeting appears to keep such mistargeting to a minimum, perturbation of the equilibrium between OMM targeting and ER mistargeting by the GET pathway can lead to aberrant accumulation of clients in the wrong membrane. Similar to nonmitochondrial TA proteins ejected from the OMM by Msp1, a parallel ATP-dependent mechanism for displacing TA proteins incorrectly introduced into the ER has recently been discovered (Fig. 4; McKenna et al., 2020; Qin et al., 2020). Structural and biochemical analyses of the ER-bound P5A-type ATPase Spf1 (yeast)/ATP13A1 (human)/CATP-8 (Caenorhabditis elegans) have identified it as a major quality control factor in the ER. Spf1 contains a substrate- binding pocket, laterally accessible from the membrane, that has been proposed to flip ER-associated proteins, promoting their release back into the cytosol or their topological rearrangement. Precisely how specificity for mislocalized/misinserted proteins is achieved remains unclear, but it is suggested that the lower hydrophobicity of non-ER TMDs may favor their dislocation. Furthermore, in cells lacking Spf1, the ergosterol content of the ER is altered to more closely resemble the OMM, implying that membrane composition may contribute to TA protein distribution and that Spf1 could also influence TA protein mislocalization by regulating the lipid composition of membranes (Krumpe et al., 2012). In yet another analogy to Msp1-mediated rejection of mislocalized TA proteins from the OMM, it is likely that mitochondrial proteins extracted from the ER may then be successfully retargeted to their desired destination. Indeed, the recently described ER–surface-mediated protein targeting pathway sets a precedent for such a route (Hansen et al., 2018).Concluding remarksThe initial momentum of the TA protein–targeting field following the discovery of the GET/TRC pathway has not abated. Recent years have seen not only a deepening mechanistic understanding of the intricacies of this pathway but also growing knowledge on its interplay with other targeting pathways and the safety nets present to ensure the fidelity of TA protein targeting. Building on the early structural analyses of individual proteins and protein domain complexes, recent advances in cryo-EM have now enabled visualization of larger complexes, such as the targeting factor–bound membrane insertase, allowing the route of TA proteins from the cytosol into the ER membrane to be anticipated. Alongside high-throughput microscopy–based screens, further analyses of TA protein targeting in cells have uncovered alternative routes that TA protein can take to reach the ER and have highlighted functional redundancies. The existence of different pathways for TA protein targeting and insertion into the ER, on the one hand, suggest a robust system with backup strategies in place in case a client protein escapes its normal targeting route, but on the other hand, emphasize the complexity of the capture process and selection of the optimal targeting pathway. In turn, these additional layers of complexity underscore the need for quality control pathways. The discovery of active removal of mislocalized TA proteins not only provides a mechanism for such quality control but perhaps also suggests a dynamic aspect to TA protein targeting wherein TA proteins within membranes can be removed and then either retargeted to their correct destination or reinserted if appropriate. Altogether, a picture emerges of an array of capture, insertion, and ejection machineries that are finely balanced in terms of substrate preferences to optimize TA protein localization within the cell.     

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

Comparison of Insulins Glargine and Degludec in Diabetic Rhesus Macaques (Macaca mulatta) with CGM Devices

Treating and monitoring type 2 diabetes mellitus (T2DM) in NHP can be challenging. Multiple insulin and hypoglycemic therapies and management tools exist, but few studies demonstrate their benefits in a NHP clinical setting. The insulins glargine and degludec are long-acting insulins; their duration of action in humans exceeds 24 and 42 h, respectively. In the first of this study''s 2 components, we evaluated whether insulin degludec could be dosed daily at equivalent units to glargine to achieve comparable blood glucose (BG) reduction in diabetic rhesus macaques (Macaca mulatta) with continuous glucose monitoring (CGM) devices. The second component assessed the accuracy of CGM devices in rhesus macaques by comparing time-stamped CGM interstitial glucose values, glucometer BG readings, and BG levels measured by using an automated clinical chemistry analyzer from samples that were collected at the beginning and end of each CGM device placement. The CGM devices collected a total of 21,637 glucose data points from 6 diabetic rhesus macaques that received glargine followed by degludec every 24 h for 1 wk each. Ultimately, glucose values averaged 29 mg/dL higher with degludec than with glargine. Glucose values were comparable between the CGM device, glucometer, and chemistry analyzer, thus validating that CGM devices as reliable for measuring BG levels in rhesus macaques. Although glargine was superior to degludec when given at the same dose (units/day), both are safe and effective treatment options. Glucose values from CGM, glucometers, and chemistry analyzers provided results that were analogous to BG values in rhesus macaques. Our report further highlights critical clinical aspects of using glargine as compared with degludec in NHP and the benefits of using CGM devices in macaques.

Diabetes is a group of metabolic diseases that cause hyperglycemia secondary to deficient insulin response, secretion, or both.⁴ Diabetes is categorized by the American Diabetes Association into 4 types: 1) type 1 diabetes mellitus, in which the pancreas is unable to produce insulin for glucose absorption; 2) type 2 diabetes mellitus (T2DM), when the body does not use insulin correctly; 3) gestational diabetes, in which the body is insulin-intolerant during pregnancy (or is first discovered then); and 4) other specific forms of diabetes in which the patient is particularly predisposed to becoming diabetic due to various comorbidities or to inadvertent induction caused by some medications.⁴ In 2018, 34.2 million (10.5%) Americans of all ages were diagnosed with diabetes.^22,23,30 Approximately 90% to 95% of Americans with diabetes have T2DM,²⁴ making T2DM the most common form of diabetes diagnosed in humans.T2DM is a multifactorial disease primarily determined by genetics, behavioral and environmental factors (for example, age, diet, sedentary lifestyle, obesity).^4,46,50,74 As a consequence of these factors, the pancreas increases insulin secretion to maintain normal glucose tolerance.⁷⁴ Over time, the high insulin demand causes pancreatic β-cell destruction, resulting in reduced production of insulin.^39,50,74 As β-cell destruction increases, hyperglycemia and T2DM develop. Insulin resistance and hyperglycemia are tolerated for a period of time^19,82,83 before clinical signs associated with T2DM develop (e.g., polydipsia, polyuria, polyphagia with concurrent weight loss).⁴ Once clinical signs develop, T2DM is most commonly diagnosed as a fasting blood glucose level (FBG) of 126 mg/dL or greater,^2,4 2-h plasma glucose value of 200 mg/d or greater during a 75-g oral glucose tolerance test,^2,4 and/or glycosylated hemoglobin (HbA1c) of 6.5% or greater.^2,4 Depending on the FBG, oral glucose tolerance test, and HbA1c results, various treatment options are recommended by the American Diabetes Association. Most importantly, lifestyle changes, including diet and exercise, are recommended as the first line of treatment, along with oral antihyperglycemic drugs such as metformin.^5,25,46 Treatment efficacy is evaluated with self-monitoring blood glucose or continuous glucose monitoring (CGM) devices.³ Human patients using CGM devices have achieved considerable reductions in HbA1c compared with patients not using them.³ As CGM devices have become more readily available, user friendly, and affordable, they have become an essential tool in managing T2DM.Similar to humans, most NHP affected by diabetes are diagnosed with T2DM.^80,83 NHP are predisposed to similar genetic, behavioral and environmental factors (e.g., age, diet, sedentary lifestyle, obesity);^{6,18,19,37,44,52,82,83} have similar pathophysiology;^38,81-83 are diagnosed via FBG,^39,83 HbA1c,^21,31,49,56 fructosamine,^20,83,87 and weight loss;^49,80,83,86 and are treated with exercise and diet modifications as a first line of treatment.^{11,19,39,53,79} Although the human and NHP conditions are similar, the treatment and management of T2DM is somewhat different, especially when NHP have restricted physical activity due to housing constraints.Previous studies indicate that daily dosing with insulin glargine achieves appropriate glycemic control in NHP.⁴⁸ Therefore, we implemented glargine, along with some diet modification, to improve glycemic control in our diabetic colony. Other noninsulin therapies, such as metformin, had been used, but compliance was low (for example, due to large pill size, unpleasant taste, etc.). However, achieving glycemic control using diet modification, insulin glargine treatment, monthly scheduled FBG, quarterly HbA1c, and regular weight monitoring was challenging in a large colony. Monthly FBG and fructosamine testing were performed due to affordability and practicality for NHP in a research setting. Given that fructosamine levels correlate with BG concentrations for the preceding 2 to 3 wk and HbA1c percentages relate to BG concentration over 1.5 to 3 mo,^49,87 HbA1C was selected over fructosamine for T2DM management in our colony. Determining which T2DM treatment and diagnostics are most effective can be difficult in large colonies of NHP. Therefore, improved treatment and management strategies would help to manage T2DM in NHP more efficiently.Insulin glargine is a long-acting insulin, with a half-life of 12 h and duration of action of 12 to 24 h in humans^40,55 and 12 h in dogs.^34,43,60 Once injected subcutaneously, insulin glargine forms a microprecipitate in the neutral pH environment, which delays and prolongs absorption in subcutaneous tissues.¹² Insulin degludec is a newer form of long-acting insulin, with a half-life of 25 h^41,63,62,77 and duration of action that exceeds 42 h in humans.^40,41,68,77 Insulin degludec forms a soluble and stable dihexamer in the pharmaceutical formulation, which includes phenol and zinc.^63,78 The phenol diffuses away, leading to the formation of a soluble depot in the form of long multihexamer chains in which zinc slowly diffuses from the end of the multihexamers, causing a gradual, continuous, and extended-release of monomers from the depot of the injection site.^63,78 Pharmacodynamic studies in humans, demonstrate that the “glucose-lowering effect” of insulin degluc⁴⁰ is evenly distributed over 24 h, allowing a more stable steady-state and improved wellbeing.⁷⁸ This approach could potentially reduce the number of hypoglycemic events and provide a less rigid daily injection schedule,⁵⁸ thus potentially making insulin degludec—compared with insulin glargine—a safer, alternative diabetes therapy.In addition to medical intervention, glycemic control is achieved through regular management and monitoring of BG. Self-monitoring blood glucose checks in humans^3,5 and glucose curves in animals¹⁰ are some of the management tools used to determine or evaluate therapy for T2DM patients. Telemetry systems like CGM devices are used to monitor interstitial glucose and have been used extensively in humans^3,17,33 and animals^{16,27,36,42,47,84,85} to monitor BG in real-time. Using CGM devices 1) reduces or eliminates the number of blood draws needed to collect FBG,⁶¹ 2) accurately assesses insulin therapy via a real-time glucose curve,^72,84,85 3) allows patients and clinicians to titrate treatment^61,73 as indicated, and 4) obtains continuous glucose data with reduced manipulation and subsequent decreased stress.^72,84,85 Therefore, CGM devices can be a safe and informative tool in monitoring spontaneous T2DM in NHP.Between 2015 and 2030, the prevalence of diabetes is predicted to increase by 54% to more than 54 million Americans affected by diabetes (i.e., diabetes mellitus types 1 and 2).⁷⁰ NHP are an essential model for human T2DM because of their similar pathophysiology, diagnostics, treatment, and management. As more people develop diabetes, novel therapies will continue to be developed. Studying new treatments and management tools in NHP can further human and NHP T2DM research to prevent the progression of T2DM and hopefully diminish projections for the number of future diabetes cases. Human medical literature, American Diabetes Association, and drug manufacturers all recommend giving equal doses (i.e., number of units/day) of long-acting insulins when changing from one long-acting insulin to degludec.^26,63,67 Therefore, we hypothesized that insulin degludec would provide effective glycemic control for rhesus macaques with T2DM when dosed at equivalent doses (that is, the same number of units/day) as insulin glargine. In addition, we hypothesized that CGM devices would provide accurate BG readings as compared with chemistry analyzer and glucometer BG readings, making it a more efficient and effective tool for measurement of BG levels in rhesus macaques with T2DM.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP’s activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

The delivery of nonmotile sperm to the embryo sac via a pollen tube is a key innovation that allowed flowering plants to carry out sexual reproduction without the need for water (Friedman, 1993; Lord and Russell, 2002). Both the speed and signal responsiveness of pollen tube growth are critical for successful fertilization (Johnson et al., 2019). The typical shape of a growing pollen tube cell protruding from a pollen grain is a cylinder with a dome-shaped tip (Geitmann, 2010). Maintaining such a typical tube shape during pollen tube growth is fundamental to support its ability for fast growth (Michard et al., 2017), and a plasticity range of tubular growth rates allows a pollen tube to optimize directional growth along its journey from the stigma to the ovule (Luo et al., 2017). The pollen tube cell extends mainly by tip growth, requiring huge amounts of secretion/exocytosis at the tip (McKenna et al., 2009; Grebnev et al., 2017). The newly secreted cell wall at the tip is mainly composed of esterified pectin, which is expandable, whereas cell wall remodeling at the lateral region (including pectin deesterification and callose deposition) limits expansion (Grebnev et al., 2017). The tip width of a growing pollen tube actually reflects the size of the secretion zone capped by an expandable membrane and cell wall, as a collective result of multiple pollen tube growth machineries (Luo et al., 2017).The tip-localized exocytosis of a growing pollen tube is supported by a spatiotemporal tightly controlled actin cytoskeleton network (Hepler, 2016). The actin cytoskeleton configuration in a pollen tube includes highly dynamic fine actin filaments in the apical and subapical regions and parallel longitudinal actin bundles in the shank region (Qu et al., 2017). Various actin-binding proteins, such as actin nucleation factors, actin-severing proteins, and actin-bundling factors, are responsible for organizing the dynamic actin cytoskeleton network (Ren and Xiang, 2007). For example, the actin-bundling proteins fimbrin and LIM (Lin-1, isl1, Mec3) domain-containing proteins function in shank-localized actin bundles in pollen tubes (Zhang et al., 2019). For another example, the actin nucleator formin (formin3 in Arabidopsis [Arabidopsis thaliana] and formin1 in lily [Lilium longiflorum]) functions in actin polymerization in the pollen tube tip (Li et al., 2017; Lan et al., 2018). The branched actin nucleator Actin-Related Protein2/3 (ARP2/3) complex is an evolutionarily conserved, seven-subunit complex consisting of the actin-related proteins ARP2 and ARP3 (Machesky et al., 1994). The ARP2/3 complex initiates the formation of branches on the side of preexisting actin filaments, locally creating a force-generating branched actin network that underlies cellular protrusion and movement (Blanchoin et al., 2000; Amann and Pollard, 2001; Molinie and Gautreau, 2018). The phenotypes of mutants in ARP2/3 in the moss Physcomitrella patens (Harries et al., 2005; Perroud and Quatrano, 2006), in Arabidopsis (Le et al., 2003; Li et al., 2003; Mathur et al., 2003; Brembu et al., 2004; Deeks et al., 2004), in maize (Zea mays; Frank and Smith, 2002), and in tomato (Solanum lycopersicum; Chang et al., 2019) demonstrated the broad importance of the ARP2/3 complex and its activation during cellular morphogenesis, including tip-growing cells. Perhaps surprisingly, in Arabidopsis, null ARP2/3 alleles are transmitted normally through pollen and there is no obvious root hair phenotype (Le et al., 2003; Djakovic et al., 2006).These cell growth machineries are tightly coordinated by multiple signaling pathways, including membrane-localized receptor kinases and Rho-of-Plants (ROP) GTPases (Li et al., 2018). The tomato pollen-specific and membrane-localized receptor kinases LePRK1 and LePRK2 mediate signaling during pollen tube growth (Muschietti et al., 1998). LePRK2 perceives several extracellular growth-stimulating factors, including a Cys-rich extracellular protein (Late-Anther-Specific52 [LAT52]), a Leu-rich repeat protein from pollen, and two pistil/stigma molecules, Style Interactor for LePRKs and Stigma-Specific Protein1 (Tang et al., 2002, 2004; Wengier et al., 2003, 2010), which increase the speed of pollen tube growth (Zhang et al., 2008b; Huang et al., 2014). LePRK2 antisense and RNA interference (RNAi) pollen tubes grow slower (Zhang et al., 2008b), consistent with a positive role for LePRK2 in regulating the speed of pollen tube growth. LePRK1 binds LePRK2 (Wengier et al., 2003), but LePRK1 plays a negative role in pollen tube growth by controlling a switch from a fast tubular mode to a slow blebbing mode (Gui et al., 2014). LePRK1 RNAi pollen tubes burst more often than wild-type pollen tubes, implicating a role for LePRK1 in maintaining plasma membrane integrity (Gui et al., 2014). An Arabidopsis paralog of these LePRKs, PRK6, also localized on the tip membrane, perceives Arabidopsis attraction cues from the female, AtLURE1s, to guide pollen tube growth (Takeuchi and Higashiyama, 2016; Zhang et al., 2017).Rho family small guanine nucleotide-binding proteins called ROPs or RACs, which can switch between a GDP-bound inactive form and a GTP-bound active form, are regulators of polar growth in pollen tubes (Cheung and Wu, 2008; Yang, 2008). In Arabidopsis, ROP1-dependent signaling controls tip growth. Active ROP1 defines a cap region in the apical plasma membrane as an exocytosis zone (Luo et al., 2017). Overexpression of ROP1 or of a constitutively active version resulted in pollen tube tip swelling (i.e. increased tip width) and slower growth (i.e. reduced tube length), while overexpressing a dominant negative version of ROP1 inhibited pollen tube growth (i.e. shorter but normal width tubes). The size of the pollen tube tip reflects the aggregate activity of membrane-associated ROP at the tip (McKenna et al., 2009; Luo et al., 2017). Tomato ROPs have been reported to be associated with the LePRK1-LePRK2 complex (Wengier et al., 2003) and therefore presumably play similar roles as the Arabidopsis homologs in pollen tube growth, yet their biological roles have not been directly investigated.Guanine nucleotide exchange factors (GEFs) activate ROPs by promoting the conversion of ROP/RAC GTPases from a GDP-bound inactive form to a GTP-bound active form. Plants possess a plant-specific ROPGEF family whose members contain a highly conserved GEF catalytic domain, the PRONE (plant-specific ROP nucleotide exchanger) domain (Berken et al., 2005; Gu et al., 2006). The intracellular portions of LePRK1 and LePRK2 interact with Kinase Partner Protein (KPP; Kaothien et al., 2005), whose Arabidopsis homologs were later shown to belong to the PRONE-type ROPGEF family (Berken et al., 2005; Gu et al., 2006). Pollen tubes overexpressing nearly full-length KPP (missing eight amino acids at the N terminus) developed swollen tips with abnormal cytoplasmic streaming and F-actin arrangements (Kaothien et al., 2005). An Arabidopsis homolog of receptor kinase, AtPRK2a (also named AtPRK2), interacts with AtROPGEF12 (Zhang and McCormick, 2007) and with AtROPGEF1 (Chang et al., 2013) to affect ROP activity. Based on the in vitro catalytic activity of full-length and truncated AtROPGEF1, an autoinhibition conferred by the C-terminal variable region was proposed (Gu et al., 2006). AtROPGEF12 was also shown to interact with the guidance receptor kinase PRK6 (Takeuchi and Higashiyama, 2016).Increased expression of full-length KPP increased the speed of pollen tube growth without significantly affecting pollen tube shape. We show biochemically that the PRONE domain of KPP does have ROPGEF activity on several class I ROPs, with highest activity on ROP4. The N-terminal domain of KPP inhibits its own GEF activity, while its C-terminal domain enhances its own GEF activity. The C-terminal domain of KPP is also required for its interactions with LePRK1, LePRK2, and an actin-bundling protein, Pollen-expressed LIM2a (PLIM2a), while the C-terminal domain alone is sufficient to bind LePRK1 but insufficient to bind LePRK2. Furthermore, self-associated KPP colocalized with the actin nucleation proteins ARP2/3 complex during pollen tube growth and enriched the membrane localization of ARP2/3 in the pollen tube. Interfering with ARP2/3 activation by coexpressing a dominant negative version of ARP2 reduced the speed of pollen tube growth and alleviated the defects caused by the overexpression of truncated KPP. CK-666, a specific small molecule inhibitor of ARP2/3 activation, canceled the promotive effect of full-length KPP on the speed of pollen tube growth. These results indicate that during pollen germination and tube growth, KPP not only links pollen receptor kinase and ROP signaling but also links the actin network to the pollen tube plasma membrane, thereby directly affecting the cellular morphology and efficiency of pollen tube growth.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Effects of Buprenorphine,Chlorhexidine, and Low-level Laser Therapy on Wound Healing in Mice

Systemic buprenorphine and topical antiseptics such as chlorhexidine are frequently used in research animals to aid in pain control and to reduce infection, respectively. These therapeutics are controversial, especially when used in wound healing studies, due to conflicting data suggesting that they delay wound healing. Low-level laser therapy (LLLT) has been used to aid in wound healing without exerting the systemic effects of therapies such as buprenorphine. We conducted 2 studies to investigate the effects of these common treatment modalities on the rate of wound healing in mice. The first study used models of punch biopsy and dermal abrasion to assess whether buprenorphine HCl or 0.12% chlorhexidine delayed wound healing. The second study investigated the effects of sustained-released buprenorphine, 0.05% chlorhexidine, and LLLT on excisional wound healing. The rate of wound healing was assessed by obtaining photographs on days 0, 2, 4, 7, and 9 for the punch biopsy model in study 1, days 0, 1, 2, 4, 6, 8, 11, and 13 for the dermal abrasion model in study 1, and days 0, 3, 6, and 10 for the mice in study 2. Image J software was used to analyze the photographed wounds to determine the wound area. When comparing the wound area on the above days to the original wound area, no significant differences in healing were observed for any of the treatment groups at any time period for either study. Given the results of these studies, we believe that systemic buprenorphine, topical chlorhexidine, and LLLT can be used without impairing or delaying wound healing in mice.

A recent retrospective analysis using a medical insurance dataset estimated that approximately 8.2 million people experienced wounds ranging from acute to chronic conditions within the particular year analyzed, and estimated that the cost of acute and chronic wound treatments ranged from $28.1 to $96.8 billion dollars.⁵² The projected rise in the number of people experiencing wounds and the cost of wound care products⁵² have made wound healing a growing area of interest in both clinical medicine and research. Wound healing is a complex process that involves many overlapping, intricate physiologic processes. Each step can have associated deviations that may lead to enhanced, altered, impaired, or delayed healing. Animal research has been used to develop a better understanding of the basic, physiologic mechanisms of wound healing. Mice are the most commonly used animal in biomedical research, and they are used to model a host of conditions, including wound healing. Despite known anatomic and physiologic differences between murine and human skin,^17,53 this species is commonly used due to their small size, ease of handling, and relatively low cost. In addition, the overlapping phases of the wound healing process are similar in mice and humans, making mice a valuable model.⁶⁵Pain is inherent to the development of wound models. Pain receptors in the skin are sensitized during the actual wounding process and during the inflammatory response that occurs immediately after wounding.¹⁹ Pain can also occur during the cleansing and treatment of wounds.¹⁹ Just as managing wound pain is critical in human patients, The Guide for the Care and Use of Laboratory Animals (the Guide)³⁰ and other federal guidelines and regulations governing the care and use of laboratory animals strongly encourages the use of analgesics for animals that experience pain and/or distress.³⁰ Pain, which can also cause stress, may evoke a persistent catabolic state and may ultimately delay wound healing.^19,28,31,43 Therefore, adequate pain control is necessary to avoid negatively affecting or altering the wound healing process.As in human medicine, opioids are commonly used to provide analgesia to research rodents. Buprenorphine, a mixed agonist-antagonist opioid,^26,54 is a common analgesic that acts as a very weak partial agonist of the mu opioid receptor and an antagonist of the κ opioid receptor.²⁶ Buprenorphine is frequently used in animals as both a pre- and post-operative analgesic. It works by binding to the opioid receptors in the skin and other tissues. This ligand-receptor binding regulates the physiologic responses of nociception and inflammation,⁷ which are key factors in the process of healing and regeneration. Buprenorphine is often used instead of full mu-opioid receptor agonist drugs, such as morphine or hydromorphone, because it has fewer systemic side effects.²⁸ Despite their common use as analgesics, reports are mixed in terms of whether opioids, as a class, delay or impair wound healing.^11,28,35,40In addition to controlling pain, minimizing wound contamination and preventing infection is critical to wound healing. The use of antiseptics is often favored over the use of antibiotics as the former presents less chance for developing antibiotic resistance.⁶ As an antiseptic, chlorhexidine is commonly used to irrigate, cleanse, and treat cutaneous wounds. Chlorhexidine has high antimicrobial activity against gram-positive and gram-negative bacteria and some fungi and viruses.⁴ Although considered to be relatively safe, reports are conflicting with regard to whether chlorhexidine delays or impairs wound healing.^4,9,50,57Laser techniques have been used medically for many years, and their powerful, but precise capabilities have rendered them a unique surgical and therapeutic modality. In brief, when the electrons of atoms move to higher energy levels, these electrons absorb energy. This excited energy state is unstable and temporary. The natural return of electrons to their more stable ground state releases energy in the form of photons or light. Light Amplification by Stimulated Emission of Radiation (LASERS) are characterized by the photon stimulation of an already excited electron. This stimulation causes the emitted light to be amplified, as demonstrated by the intense, bright light that is emitted from lasers.⁶³ The concept of low-level laser therapy (LLLT) has garnered interest as a therapeutic modality in both human and veterinary medicine. Specifically characterized as laser therapy using a low power output and a low power range, LLLT is distinguished from other forms of laser therapies by certain parameters such as wavelength, pulse rate and duration, total irradiation time, and dose.⁴⁴ Although the mechanism of action for LLLT is not completely understood,^46,64 the absorption of red and near infrared light energy may reduce detrimental, inflammatory substances^13,15,24,56 while simultaneously stimulating restorative processes.^15,24,46,64 The reduced photothermal impact of LLLT⁴⁴ is reported to produce beneficial physiologic and biologic effects including analgesia, reduction in inflammation, and acceleration of healing.⁴⁸ The initial report of LLLT as a therapeutic modality found accelerated wound healing and fur regrowth in mice exposed to LLLT.^13,44,46,64 LLLT has since been used as a sole or adjunct therapy for a variety of conditions including tooth root resorption,⁵⁵ traumatic brain injuries,⁵⁸ and tendon, muscle, and bone injuries.^2,3,25,38Studies conducted to assess the effects of LLLT on healing often use parameters of normal wound healing to analyze how LLLT influences those parameters in comparison to healthy, undamaged tissue and damaged tissue not receiving laser therapy. Despite the numerous studies designed to investigate the effects of LLLT on wound healing, conflicting reports exist regarding its efficacy.^{15,17,46,22,23,24,29,34,38,39,55,56,60,64} A recent study in dogs reported accelerated healing and improved cosmetic appearance of a hemilaminectomy surgical site after LLLT,⁶⁰ while other canine studies reported no significant differences in the healing of surgically induced skin wounds between dogs that did and did not receive LLLT.^22,34 Similarly, in an attempt to study the effects of LLLT in pigs, an animal with skin very similar to that of humans, no significant differences were reported in the healing of surgically created skin wounds between swine that did and did not receive LLLT.²⁹ Studies using diabetic rats with excisional cutaneous wounds reported accelerated wound healing,^17,46 and beneficial results were reported in a similar study using diabetic mice.^56,64 While fewer studies have been conducted on the use of LLLT in rodents without concomitant comorbidities, LLLT has been reported to accelerate wound healing in healthy rodents.^15,24 Conversely, some studies found that LLLT does not accelerate or significantly improve wound healing in rodents.^24,39We performed 2 separate studies to investigate the effects of a commonly used opioid, a topical antiseptic solution, and LLLT on excisional wound healing in mice. At the time the initial study (study 1) was conducted, some of our investigators were reluctant to use the recommended analgesic, buprenorphine, due to concern about interference with their study outcomes. Therefore, we conducted study 1 to determine if a single dose of peri-operative buprenorphine would delay healing of a full-thickness excisional wound or a partial-thickness felt wheel dermal abrasion. We also examined the effects of topical chlorhexidine solution on wound healing. The chlorhexidine concentrations used in study 1 were prepared using our standard operating procedure at that time. Study 2 was conducted after study 1, with the design expanded to evaluate a sustained release buprenorphine formulation and LLLT. Study 2 used a full-thickness excisional biopsy to determine the effect of LLLT on excisional wound healing. Commonly used doses of systemic Buprenorphine Sustained Release (SR) and topical chlorhexidine were also included to evaluate their effect on excisional wound healing. The concentration of chlorhexidine in the revised, approved standard operating procedure had been decreased due to literature suggesting that higher concentrations may inhibit healing.^4,49,61 For both studies, we hypothesized that the use of buprenorphine and chlorhexidine would have no effect on the rate of wound healing, and that LLLT would accelerate wound healing in a full-thickness excision as compared with a control.     

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 10⁶, 10⁷ or, 10⁸ CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 10⁷ or 10⁸ CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16.h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Protein binding is an important component of pharmacokinetic/pharmacodynamic (PK/PD) research. In vitro measurement of protein drug binding is an essential component of the research and development of novel drugs. However, in vitro studies often poorly mirror the in vivo condition.^9,42 Pharmacokinetic studies early in drug development provide a means to assess the time course of drug effects in the body and drug distribution and availability.⁴² From a PK/PD modeling perspective, protein binding is an important factor in the kinetics and dynamics of drug availability in vivo.^21,35,36,40 These complex relationships are used to project efficacious doses in humans and take into consideration differences in plasma protein binding between preclinical species and humans.^8,44A variety of acute phase proteins (APP) exist across all species and increase in response to inflammatory, infectious and traumatic events.^{5,9,12,13,19,21,22,29,45,53} APPs are potential biomarkers for detection and monitoring of various disease states including cancer.^{2,18,24,34,39,40,47,50,52} Because of this, enhanced understanding of drug binding characteristics to APPs early in the development phase will promote the design of more efficacious therapeutics. Alpha-1 acid glycoprotein (AGP), a ubiquitous major APP that is present in rats,^9,46 has significant drug binding properties and binds to many basic and neutral compounds. Normal AGP levels in plasma of naïve rats range from 0.1 to 0.32 mg/mL.⁴⁴ The importance of AGP as related to drug discovery and development will be bolstered by greater understanding of the sources of AGP stimulation in established animal models. For example, AGP modulates the immune response in a rodent shock model in which it is thought to maintain normal capillary permeability to ensure perfusion of vital organs.^30,33 In addition, elevated AGP levels are present in animal models of infection and inflammation.^{11,20,27,32,41,48}In surgically modified animals, AGP levels may be elevated after surgical manipulation, which unavoidably induces local transient inflammatory responses.^8,25,51 In addition, infections may develop postoperatively leading to increased AGP levels. Chronic catheterization has been linked to increased incidence of infection.^3,8,37 Surgically modified animals should not be placed on study if aseptic technique was not adhered to during surgical preparation and instrumentation.^6,37 Contamination may occur within or at the external portion of a catheter, usually resulting in more obvious signs of infection. Routine PK studies in rats involve implantation of vascular catheters through which drugs are administered and blood samples are taken over time. Catheterized animals are typically perceived as being healthy and thus are enrolled in and remain on study unless they develop obvious clinical signs of infection or illness. However, an occult infection may be present even with a patent catheter. As such, understanding the direct effect of subclinical infection in modulating AGP levels and drug binding is critical, as AGP levels may affect drug levels in study animals with persistent subclinical infection. In this event, the PK data generated may be altered due to selective binding to AGP, thus confounding data interpretation.A possible application of AGP is its potential utility as a biomarker for evaluating health status animals in drug development. The use of AGP as a select biomarker for monitoring and identifying sick animals and/or predicting the potential impact of subclinical infection on drug PK/PD is highly desirable. A screening tool such as this could help to optimize animal selection by reliably identifying healthy animals. Improved intra-study health monitoring would promote confidence in PK/PD data and its predictive value.The focus of this research was to develop a sensitive, reliable and reproducible model of subclinical infection in the rat using the ubiquitous skin contaminant, S. aureus. We selected AGP as a biomarker that would promote health status screening and enhance PK/PD characterization of AGP binding drugs (that is basic and neutral) in the presence or absence of subclinical infection. The model was validated by evaluating the impact of increased AGP levels on propranolol, a drug known to have high binding affinity to AGP.^{4,7,10,26,28,31,49} Ultimately, establishing this model will provide heightened visibility of the protein binding characteristics of drugs and yield more informed data interpretation.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

Indicators of Postoperative Pain in Syrian Hamsters (Mesocricetus auratus)

Despite the use of Syrian hamsters (Mesocricetus auratus) in research, little is known about the evaluation of pain in this species. This study investigated whether the frequency of certain behaviors, a grimace scale, the treat-take-test proxy indicator, body weight, water consumption, and coat appearance could be monitored as signs of postoperative pain in hamsters in a research setting. Animals underwent no manipulation, anesthesia only or laparotomy under anesthesia. An ethogram was constructed and used to determine the frequencies of pain, active and passive behaviors by in-person and remote videorecording observation methods. The Syrian Hamster Grimace Scale (SHGS) was developed for evaluation of facial expressions before and after the surgery. The treat-take-test assessed whether surgery would affect the animals’ motivation to take a high-value food item from a handler. The hypothesis was that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups. At several time points, pain and passive behaviors were higher than during baseline in the surgery group but not the anesthesia-only and no-intervention groups. The SHGS score increased from baseline scores in 3 of the 9 animals studied after surgery. The frequency of pain behaviors and SHGS scores were highly specific but poorly sensitive tools to identify animals with pain. Behaviors in the pain category were exhibited by chiefly, but not solely, animals that underwent the laparotomy. Also, many animals that underwent laparotomy did not show behaviors in the pain category. Treat-take-test scores, body weight, water consumption, and coat appearance did not change from baseline in any of the 3 groups. Overall, the methods we tested for identifying Syrian hamsters experiencing postoperative pain were not effective. More research is needed regarding clinically relevant strategies to assess pain in Syrian hamsters.

Pain experienced by laboratory animals can affect both animal welfare and research results. Little is known about the evaluation of pain in Syrian hamsters (Mesocricetus auratus) in the laboratory setting. However, various research models using Syrian hamsters involve surgery and are presumed to cause pain.^16,47,49 In 2018 alone, the USDA reported that 35,695 hamsters were used for research studies involving painful procedures.⁴⁸ Previously published behaviors exhibited by hamsters in response to pain include hunched posture with head down, reluctance to move, increased depression or aggression, extended sleep periods, and weight loss.^7,8,10,16,21 How these behaviors are affected by factors such as the type of painful stimulus, anesthetic protocol, handling procedures, and environmental conditions is unclear. The practicality of observing these signs in the research environment is uncertain and likely complicated by the nocturnal nature of Syrian hamsters and an assumed propensity of this species to mask pain, much like other prey species.^8,14,16A significant need exists for published data investigating whether behavioral observations or other clinical indicators can help recognize, quantify, or monitor pain in hamsters in a research setting. Detailed behavioral observations and well-controlled studies are needed to develop a system to assess postoperative pain in laboratory animals.^8,33 Moreover, little information is available on the efficacy of analgesic agents in hamsters.¹ The few studies of analgesics in hamsters rely on the mitigation of evoked pain responses (such as using a hot plate), which has limited relevance to clinical situations such as postoperative pain.^8,32,36,51 To date, no published literature has evaluated the efficacy or safety of analgesics to treat postoperative pain in hamsters. Validated real-time and practical methods for evaluating pain in Syrian hamsters would support the evaluation of analgesic efficacy in this species.Various assessments have been developed to identify signs of pain in other species. Behavioral ethograms have been used to evaluate pain and analgesic efficacy in mice, rats, rabbits, and guinea pigs in the research environment.^{5,6,20,23,25,34,35,39-41,53} Another tool used to evaluate pain in animals is the grimace scale, which has been developed for mice, rats, rabbits, ferrets, cats, sheep, pigs, horses, and even harbor seals.^{3,4,9,11,13,15,19,22,26,30,37,45,50} The use of a proxy indicator, such as burrowing and time-to-integrate-to-nest in mice and time-to-consume in guinea pigs, can be used as an additional tool for the evaluation of pain.^{5,17,18,35,38}Because none of the previously mentioned assessment techniques were specific to hamsters, we here explored using these approaches to detect pain in Syrian hamsters that underwent laparotomy in a laboratory setting. We developed a species-specific ethogram and the Syrian Hamster Grimace Scale (SHGS). We also devised a novel proxy indicator of pain for use in Syrian hamsters, the treat-take-test (TTT), which is based on hamsters’ natural behavior to hoard food.^16,46,49,52 Although water intake, body weight, and coat appearance are non-specific indicators of pain, we also measured these parameters.^5,19,23,33 Furthermore, we analyzed the effects of the presence of an observer and time of day. We hypothesized that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups.     

Comparative Efficacy of Two Types of Antibiotic Mixtures in Gut Flora Depletion in Female C57BL/6 Mice

Over the last decade, interest in the role of the microbiome in health and disease has increased. The use of germ-free animals and depletion of the microbial flora using antimicrobials are 2 methods commonly used to study the microbiome in laboratory mice. Germ-free mice are born, raised, and studied in isolators in the absence of any known microbes; however, the equipment, supplies, and training required for the use of these mice can be costly and time-consuming. The use of antibiotics to decrease the microbial flora does not require special equipment, can be used for any mouse strain, and is relatively inexpensive; however, mice treated in this manner still retain microbes and they do not live in a germ-free environment. One commonly used antibiotic cocktail regimen uses ampicillin, neomycin, metronidazole, and vancomycin in the drinking water for 2 to 4 wk. We found that the palatability of this mixture is low, resulting in weight loss and leading to removal of mice from the study. The addition of sucralose to the medicated water and making wet food (mash) with the medicated water improved intake; however, the low palatability still resulted in a high number of mice requiring removal. The current study evaluated a new combination of antibiotics designed to reduce the gut microbiota while maintaining body weights. C57BL/6NCrl mice were placed on one of the following drinking water regimens: ampicillin/neomycin/metronidazole/vancomycin water (n = 16), enrofloxacin/ampicillin water (n = 12), or standard reverse osmosis deionized water (RODI) (n = 11). During an 8 day regimen, mice were weighed and water consumption was measured. Feces were collected before and after 8 d of treatment. Quantitative real-time PCR (real-time qPCR) for 16S bacterial ribosome was performed on each sample, and values were compared among groups. The combination of enrofloxacin and ampicillin improved water intake, together with a greater reduction in gut flora.

Interest in the intestinal microbiome and its role in human health has increased dramatically over the last decade. The microbiota has been implicated in metabolic, infectious, and inflammatory disease, and its role has been investigated not only in the gut,^2,8,18,38 but also in vasculature,^5,6,19,39 kidney,¹³ liver,²⁸ lung,^9,34,37 and brain.^12,15 Animal models have been important in furthering our understanding of the microbiota. Two approaches to studying microbiota in mice are the use of germ-free mice^22,35,42 and depletion of the flora with oral administration of antibiotics.^12,17,18 Both approaches have advantages and disadvantages. Germ-free mice are bred in isolators and are free of microorganisms from birth, allowing studies in mice with no microbes present; mice can then be used to generate gnotobiotic mice in which only known microbes are present. However, to remain germ-free, mice must be maintained in isolators under aseptic housing conditions, which is both costly and labor intensive. In addition, alterations of microbiota in early life may cause sustained effects on body composition¹⁰ and lasting negative consequences on the host immune system.³¹ A more economic approach has been to deplete mouse gut microflora using a combination of broad-spectrum antibiotics given either by oral gavage or in the drinking water. The primary limitation with antibiotic treatment of mice is that not all microbes are eliminated; which can potentially make reproducibility in certain types of studies such as those involving microbial transplantation²⁹ very difficult. However, antibiotic-induced gut dysbiosis can be used on conventionally raised mice without the limitations imposed by maintaining a sterile living environment. Direct handling of the mice is possible, allowing behavioral and imaging assessments, which are not be feasible for mice housed in isolators. Several broad-spectrum antibiotic treatment regimens in the drinking water have been used for gut microbe depletion.^{7,16,20,25-27,41} One of the more commonly used combinations is comprised of 4 antibiotics (ampicillin, neomycin, metronidazole, and vancomycin) added to the drinking water for periods ranging from 1 to 4 wk.^{5,9,13,19,23,30,32,34} This cocktail is effective at depleting gut microbes; however, a previous study in our laboratory found it to be highly unpalatable. Dehydration and weight loss can occur in mice receiving antibiotics in the drinking water, and the magnitude of the effect can be significant, depending on the mouse strain.^21,30,33 The weight loss can result in a substantial number of mice being removed from studies due to animal welfare concerns as reported in a previous study in which 5 of 5 mice given ampicillin, neomycin, metronidazole, and vancomycin reached eighty percent of baseline body weight and were subsequently removed.³³ A reduction in water consumption is also likely to interfere with effective antibiotic treatment and may prolong the time necessary to achieve adequate microbial depletion. Palatability enhancers such as glucose,^5,13 sucrose,⁴¹ and flavored water²³ are sometimes combined with the antibiotics in drinking water. The aim of the current study was to determine whether 8 days of treatment with an alternative mixture comprised of 2 antibiotics (enrofloxacin and ampicillin) was sufficient to deplete the gut flora as compared with the widely used combination of ampicillin, neomycin, metronidazole, and vancomycin. We hypothesized that the combination of 2 antibiotics would be at least equivalent to the combination of 4 antibiotics in reducing the gut flora while causing less weight loss.