extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
The first-in-class alkylating HDAC inhibitor EDO-S101 is highly synergistic with proteasome inhibition against multiple myeloma through activation of multiple pathways

The first-in-class alkylating HDAC inhibitor EDO-S101 is highly synergistic with proteasome inhibition against multiple myeloma through activation of multiple pathways

EDO-S101 is a first-in-class alkylating, histone deacetylase inhibitor (HDACi) fusion molecule with dual activity. It structurally combines the strong DNA damaging effect of bendamustine with a fully functional pan-HDAC inhibitor, vorinostat (Supplementary Figure S1) that is intended to simultaneously deliver DNAdamaging activity while inhibiting DNA repair activity, and is currently undergoing phase I clinical testing in hematologic malignancies. 1 EDO-S101 has shown a strong preclinical activity in vivo against multiple myeloma (MM), leukemia and B-cell lymphomas in preclinical models with a toxicity profile similar to bendamustine. 2 EDO-S101 has remarkable activity in vivo in the multi-drug resistant Vk12653 transplant model of relapsed/ refractory MM, in which it was the only drug identified with single agent activity. 3 Bendamustine has substantial activity against B-cell malignancies and vorinostat sensitizes the same type of cancers against alkylators or proteasome inhibitors (PI). 4,5 Cytotoxicity of PI in MM relies on excess induction of proteotoxic stress and triggering of the unfolded protein response (UPR) 6 upon proteasome inhibition, and HDACi synergize with PI by interfering with the α-tubulinmediated transport of polyubiquitinated proteasome substrates to lysosomal destruction. 7 Thus, combining EDO-S101 with PI is expected to deliver mechanism-based, highly synergistic cytotoxicity, based on combining proteasome inhibition with histone deacetylase inhibition and alkylating activity in one molecule. We here focus on the preclinical in vitro exploration of the potential use of EDO-S101 in combination with PI against MM and B-cellderived malignancies.
EDO-S101 in combination with PI bortezomib induced the strongest cytotoxic effect compared with vorinostat or bendamustine alone or their combination with bortezomib ( Figure 1a). Further, EDO-S101 showed superior cytotoxicity in MM in vitro compared to melphalan, cyclophosphamide or bendamustine (Supplementary Figure S2A), showed alike cytotoxicity compared to vorinostat+bendamustine combination (Supplementary Figure S2B) and induced more effective synergistic cytotoxicity in combination with bortezomib and second generation PI carfilzomib, compared to bendamustine or melphalan (Supplementary Figure S2C). Likewise, EDO-S101 induced robust synergistic cytotoxicity in combination with all approved proteasome-inhibiting drugs, as well as PI in clinical development (Supplementary Figure S3, Supplementary Table S1). This synergy was observed already at a 4 μM drug concentration of EDO-S101 and yielded highly significant combination indices for both bortezomib and carfilzomib in a variety of MM cell lines, cell lines from hematologic (mantle cell lymphoma, ABC-type and GC-type diffuse large B-cell lymphoma, acute myeloid leukemia) and nonhematologic malignancies, as well as primary cells from hematologic cancers (Supplementary Figures S4-S6; Supplementary Table S2). The combination between EDO-S101 and carfilzomib was synergistic to overcome bortezomib-resistance, as shown using the AMO-BTZ model previously described 8 Figure S4A).

(Supplementary
The antineoplastic activity of HDAC inhibitors is partly mediated through modulation of nuclear histone acetylation, which results in epigenetic changes, and partly through the modulation of protein acetylation in the cytosol, which functionally controls the activities of several key proteins involved in basic cellular functions as well as oncogenesis. 9 Acetylation of α-tubulin controls the transport of polyubiquitinated protein to aggresomal proteolysis and is a major molecular mechanism for the cytotoxic synergy between proteasome inhibitors and HDAC inhibitors. 7 EDO-S101 resulted in superior histone acetylation compared to vorinostat, as revealed by acetylated H3K9, and in particular induced robust acetylation of α-tubulin, also in contrast to vorinostat, suggesting superior inhibition of aggresomal transport of poyubiquitinated protein ( Figure 1b). Consistent with this, we observed increased accumulation of polyubiquitinated protein in cells treated with EDO-S101, compared to controls treated with vorinostat or untreated controls. Combined treatment with bortezomib and EDO-S101 led to a maximum increase in cellular polyubiquitinated protein, highlighting the synergy between both pathways. We can exclude a direct effect of EDO-S101 on proteasome activity because EDO-S101 treatment did not change activity-based labeling of active proteasome subunits in treated, viable cells, in contrast to bortezomib (Supplementary Figure S7).
The activation status of the UPR, and in particular the activation levels of IRE1/XBP1 determine proteasome inhibitor sensitivity of MM. 10,11 EDO-S101 caused phosphorylation of IRE1, the key activator of the UPR, already 1 h post treatment (Figure 1c). The combination of EDO-S101 with bortezomib resulted in more effective IRE1 activation than either agent alone, as evidenced for example, by the induction of CHOP expression already after 1 h (Figure 1c and Supplementary Figure S8C). IRE1 activation leads to splicing and activation of the XBP1 transcription factor, which significantly increased 4 h post treatment with EDO-S101 or bortezomib treatment alone and further increased upon treatment with the combination of EDO-S101 with bortezomib (Supplementary Figure S8A). By contrast, vorinostat at the same dose did not induce IRE1/XBP1 activation. In summary, treatment with EDO-S101 and bortezomib resulted in robust activation of the UPR, as evidenced by induction of binding immunoglobulin protein (BIP) and protein disulfide-isomerase (PDI) and triggered the ATF4/CHOP signaling pathway that connects proteotoxic stress with the induction of apoptosis (Figure 1c and Supplementary Figure S8B and D). These effects were significantly stronger after combination treatment, compared to either agent alone.
Given the accumulation of polyubiquitinated protein upon simultaneous inhibition of the proteasomal and aggresomal pathways after combination treatment with bortezomib and EDO-S101, we hypothesized that this might activate alternative cellular proteolytic systems, in particular autophagy. Indeed, accumulation of MAP1LC3A/B protein after EDO-S101 treatment, alone and in particular in combination with proteasome inhibition, indicated activation of autophagy upon such dual inhibition of proteasomal and aggresomal protein disposal (Figure 1d).
The alkylating activity of EDO-S101 presumably leads to alkylation of DNA and subsequent double strand breaks, while histone acetylation triggers chromatin de-condensation leading to activation of transcription of cell cycle inhibitors, which both are predicted to result in cell cycle arrest. 12 EDO-S101 alone caused marked S-phase arrest 8 h post treatment that was further potentiated in combination with bortezomib ( Figure 1e). Neither vorinostat nor bendamustine alone or in combination with bortezomib showed significant decrease in cell cycling. Bortezomib-induced cytotoxicity involves upregulation of the CDKN1A (p21) inhibitor of cell cycle via inhibition of p21 degradation. 13 EDO-S101 alone likewise caused a marked increase in p21 expression already 1 h post treatment, which was further increased during the treatment with a combination of EDO-S101 In all experiments, cells were exposed to the indicated drug concentrations for 1 h, followed by removal of the drugs and subsequent incubation in drug-free medium for the indicated time points. Statistically significant differences from untreated controls are marked with asterisks; *Po0.05; **P o0.01; ***P o0.001.
Letter to the Editor and bortezomib (Supplementary Figure S9), also in contrast to vorinostat. Conversely, EDO-S101 more effectively reduced CDKN1B (p27) levels alone and in combination with bortezomib, compared to vorinostat (Supplementary Figure S9). Thus EDO-S101 synergizes with proteasome inhibition to induce cell cycle arrest in MM cells.
Overexpression and aberrant activation of c-MYC prevents apoptosis and drives B cell-derived malignancies, including in particular aggressive lymphoma and MM. 14 EDO-S101 reduced c-MYC expression by 70% already after 4 h, in contrast to vorinostat. The combination of EDO-S101 with bortezomib led to more than 90% reduction in c-MYC expression (Supplementary Figure S10A).
UPR-induced cytotoxicity involves the mitochondrial apoptotic pathway and shifts the balance between pro-apoptotic and antiapoptotic signals. 15 EDO-S101 induced expression of proapoptotic NOXA (BH3-only BCL2 family member) already 2 h post treatment, while the largest effect was observed 4 h post treatment (Figure 1f and Supplementary Figure S10B). Bortezomib likewise induced NOXA expression, and the combination of EDO-S101 with bortezomib had a more pronounced signaling effect than either agent alone. Consistent with this, EDO-S101 resulted in downregulation of anti-apoptotic BCL2 on protein and transcript levels, and phospho-BCL2 was virtually absent from cells treated with EDO-S101 alone or in combination with bortezomib ( Figure 1f and Supplementary Figure S10C). The induction of apoptotic signaling by EDO-S101, alone and in particular in combination with bortezomib, was further supported by an increased ratio of BAX/BCL2 (Supplementary Figure S10D), and was associated with a decrease in the anti-apoptotic effector phospho-MCL1 and, importantly, cleaved PARP (Figure 1f). Strikingly, EDO-S101 alone at 40 μM pulse treatment resulted in 70% apoptotic cells, which increases to 90% when combined with bortezomib 100 nM, while neither equimolar bendamustine nor vorinostat significantly increased the fraction of cells with an apoptotic phenotype by flow cytometry over background levels (20%, Figure 1g).
In summary, EDO-S101 is the first representative of the A-HDAC principle, a new approach in chemotherapy that chemically combines an alkylating agent with a pan-histone deacetylase inhibitor to simultaneously damage DNA and block damage repair. We here show that EDO-S101 has in addition potent ER stress-inducing, HDAC6-inhibiting, immunomodulatory, cell cycleinhibiting, pro-apoptotic and c-MYC-antagonistic activity, in contrast to vorinostat or bendamustine ( Figure 2). This is in line with the superb synergistic cytotoxic activity of EDO-S101 in combination with proteasome inhibitors against MM and a wide variety of B-cell neoplasms in vitro, as well as with the activity of EDO-S101 in a predictive in vivo model for relapsed/refractory MM. Based on our data, safety and activity of EDO-S101 should be assessed as a potential next-generation alkylating drug with dual, histone-acetylating activity, for multiple myeloma and B-cell malignancies, in particular in combination with proteasome inhibitors. Figure 2. Schematic presentation of EDO-S101 DNA alkylating and HDAC inhibitory role and synergistic effect with bortezomib, and the consequences for the cell. EDO-S101 causes DNA alkylation and histone/protein acetylation due to histone deacetylases (HDAC) inhibitory activity. DNA alkylation leads to the induction of double strand breaks in the DNA, which is potentiated by the vulnerability of the DNA to alkylation due to opened chromatin structure and increased transcriptional activity. The DNA damage and increased transcription of cell cycle inhibitors together with inhibition of cell cycle inhibitors (p21) degradation by bortezomib leads to S-phase arrest. Further, transcriptional activation leads to accumulation of polyubiqitinated proteins dedicated for proteasomal degradation which in turn is blocked by bortezomib. Accumulation of polyubiquitinated proteins causes ER stress which unresolved leads to apoptosis. Intracellular effects are summarized in the table below.


s2fieldsofstudy:
Biology
Environmental Science

text:
New record of a phoretic flea associated with earwigs (Dermaptera, Arixeniidae) and a redescription of the bat flea Lagaropsylla signata (Siphonaptera, Ischnopsyllidae)

Abstract Lagaropsylla signata (Wahlgren, 1903), previously known only from the Island of Java, Indonesia is redescribed and reported for the first time in Deer Cave, Gunung Mulu National Park, Sarawak, Malaysia (west coast of Borneo). Many were found clinging to the earwig Arixenia esau Jordan, 1909. A similar account of a phoretic flea (Lagaropsylla turba Smit, 1958) on the same species of cave-dwelling earwig has been reported in peninsular Malaysia in a well-documented association with the hairless naked bulldog bat, Cheiromeles torquatus Horsfield, 1824. The association of Lagaropsylla signata with Arixenia esau is parallel to the evolution and co-existence with bats in Deer Cave just as in the case of Lagaropsylla turba, Arixenia esau, and Cheiromeles torquatus. The evidence suggests that Lagaropsylla turba and Lagaropsylla signata are obligate phoretic parasites whose survival depends on Arixenia esau to access a bat host. Arixenia esau is reported for the first time in Deer Cave and the occurrence of Lagaropsylla signata on the island of Borneo represented a new record, previously being found only on the island of Java. Images of Lagaropsylla signata attached to Arixenia esau are provided. Xeniaria jacobsoni (Burr, 1912), often associated with Arixenia esau in other geographical areas, was not present in the material examined from Deer Cave. The natural history of the earwig genera Arixenia Jordan, 1909 and Xeniaria Maa, 1974 are discussed and summarized relative to their associations with phoretic fleas and their bat hosts.

Introduction
Phoresy occurs in some insects and arachnids in which one species attaches to another species in a commensal relationship for the purpose of increasing their ability to disperse from one place to another. There is seldom a detrimental effect on the transporting host species. In some cases, attachment to another species is accidental, while others have evolved into vital components of their life history. Although there is an account of a bird flea attaching to a wasp (Rothschild & Clay, 1952), this was attributed to an accidental association of a wasp foraging on a flea-infested avian carcass. Lagaropsylla turba Smit, 1958 is the only documented species of flea that truly demonstrates phoretic behavior for which there was an association between a flea, another insect (A. esau Jordan, 1909), and a bat host (the naken bulldog bat Cheiromeles torquatus Horsfield, 1824). In this scenario, an earwig provides a vehicle for L. turba to come into contact with its only known host, C. torquatus. Nakakta and Maa (1974) provided a summary of the commensal forms of the dermapterid suborder Arixeniina and their associated molossid bat species. Important works cited by Nakata and Maa (1974) included: Jordan (1909), Jacobson (1912), Burr and Jordan (1913), Audey (1952), Cloudsley-Thompson (1957), Medway (1958), Giles (1961), and Popham (1962). Medway (1958) and Marshall (1977) described some crucial bionomical inter-relationships relative to L. turba, A. esau, and C. torquatus, while Marshall (1981) discussed similar parallel behaviors of co-existing associations between bird lice, hippoboscids, and their avian hosts. We reported herein a second flea species [Lagaropsylla signata (Wahlgren, 1903)] that has a similar phoretic association with A. esau. We also redescribed L. signata and further documented a new locality for both L. signata and A. esau.

Materials and methods
Earwigs were collected from the floor of Deer Cave while conducting a general insect survey of Gunung Mulu National Park in October 2006 (04°01'18"N, 114°49'24"E) (KBM & GJS) and in January 2009 (04°02'32.8"N, 114°48'49.6"E) (GJS). Specimens were collected in the same location deep within the main gallery of the cave in the early evening. No earwigs were found in the entrance area and path through the first half of the cave, although mixed rock and guano piles were present. Approximately 1000 m and within sight of the "Garden of Eden doline" (a cave ceiling collapse that allows light to enter the main gallery), the path climbed an isolated, cone-shaped hill. Very few earwigs were present at the base of this hill, but numbers increased with elevation. The top of the hill, the observation path, and railings included earwigs. Thousands were present in October 2006 and only a few were present in January 2009. Individuals were observed actively walking, mating, and resting in small cracks. Males, females and nymphs were collected using forceps to overcome their strong grip on the surface of rocks, railings, and clothing. The greatest concentrations of individuals were at the highest points on the hilltop, which suggests a negative geotactic behavior. Individuals were not observed in the area around the base of the hill and nearby cave walls.
All specimens were collected into and stored in 95% ethanol and later examined in the laboratory. During microscopic examination, many fleas were noted attached to the earwigs. One species of earwig was present and the mouthparts and genitalia were dissected to facilitate and confirm our identification. Earwigs were photographed in ethanol with the aid of a Canon 6D DSLR camera and Visionary Digital Passport II imaging system. Image stacks were montaged with Zerene Stacker v.1.04. Fleas were mounted on microscope slides in accordance with procedures outlined by Hastriter andWhiting (2003:1043)
Diagnosis. Both sexes may be distinguished from all other species of Lagaropsylla by a narrow band separating the margin of the frons from a white area anterior to the frontal row of fine setae. This band is hardly wider than that of the marginal layer of the frons (Figs 1-2). Closely allied to Lagaropsylla mera mera Jordan and Rothschild, 1921,   Description. Walgren (1903) provided only a brief description of L. signata. A detailed description is therefore provided to include illustrations of the species. Unless otherwise specified, counts of spines and setae are for one side of the flea.
Head. Frons evenly rounded with very thin band layered between margin and a broader white area caudad; caudal margin of white zone lined with a dozen small setae from oral angle to upper antennal fossa. Second genal tooth longer than first. Pre-oral tuber short and thick, only half the length of first genal tooth. Eye fused into upper heavily sclerotized margin of genal lobe, hardly discernible as distinct eye. Labial palpus of five short segments; sub-equal to length of maxillary palpus. Occipital area with dorsal incrassations (Figs 1-2).
Thorax. Length of pronotum equal to height of pronotum; 18 sharp ctenidial spines (both sides) equal to length of pronotum. One dorsal incrassation in pronotum; two dorsal incrassations in meso-and metanota. Prosternosome with antero-ventral area expanded ventrad. Pleural rod fused in center of sclerotic dome. Mesosternum and mesoepimeron fused as one; with two tuberculiform sclerotizations at juncture of sternum and epimeron. Pleural ridge feebly developed; pleural arch lacking. Lateral metanotal area dorso-ventrally flattened. Metepimeron with 12 setae in male and 21 or 22 in female (Figs 1-2).
Legs. Oblique suture of mesocoxa only indicated on ventral margin. Notch in metacoxa vestigial. All femora lacking lateral or mesal setation. Dorsal margin of all tibiae with six dorsal notches. Distitarsomeres each with five pairs of lateral plantar bristles; most proximal pair set onto plantar surface between second pair (Figs 8-9).
Unmodified abdominal segments. Abdominal terga I-VII each with a dorsal incrassation. Main rows of setae on T-I-VI interrupted; one seta below level of each spiracle. Spiracles round. One long antesensilial bristle. Sternum II of male without lateral setae. Female S-II with lateral patch of 14 setae; some short and others long and slender (Fig. 2). Both male and female with vertical parallel reticulations on S-II. Male without setae on S-III; one row of setae on S-IV-VII (2, 3, 3, 3). Female with one row of setae on S-III-VI (6, 4, 4, 4).
Modified abdominal segments, male. Saddle of T-IX and manubrium forming an obtuse angle. Basimere more convex on ventral margin than dorsal margin. Basimere with two or three small setae along dorsal margin; two moderately stout setae at apex. Acetabulum of telomere placed approximately midway between base and apex of basimere. Telomere half the length of basimere; slightly angled at ventral apical third terminating as acute angle at apex. Telomere with five or six minute setae along ventral margin; one minute seta at apex and two minute setae on dorsal margin (Fig. 3). Tergum VIII vestigial. Tendon of S-IX long; curved beyond and over apex of aedeagal apodeme (Fig. 5). Left and right halves of S-VIII fused along ventral margin; with dorsal lobe encompassing aedeagus. Two short and two longer setae at juncture of fused sclerites; lateral group of six or seven small setae (Fig. 5). Distal arm of S-IX (DA9) as in Fig. 5; apex with two setae, one slightly spiniform. Ventral margin of apical portion of DA9 with three minute marginal setae. Apex of proximal arm fused with base of aedeagal apodeme (fused area inseparable with dissection). Penis rods sub-equal in length to tendon of S-IX. Dorsal margin of aedeagal apodeme convex; apex acutely terminated. Dorsal spur present; fused with dorsal surface of T-IX. Virga ventralis thick but not darkly sclerotized. Crescent sclerite nearly vertical relative to longitudinal axis of aedeagal apodeme. Median dorsal lobe rounded; distal half more lightly pigmented than that of the surrounding area. Sclerotized inner tube shorter on ventral apical margin than dorsal margin; latter appearing in lateral view as fine, hair-like extension. Dorsal armature on dorsal margin of sclerotized inner tube thorn-like. Ventral surface with similar heavily sclerotized ventral armature. Crochet thin and tapering to apex; peg-like paxillus near ventral base of crochet (Fig. 4).
Modified abdominal segments, female. Tergum VIII with two small setae near spiracle VIII, three or four long lateral setae, and four or five mesal marginal setae (two stout, two or three fine). Sternum VIII tube-like without setae. Caudal margin of S-VII with broad, truncate lobe with slight concavity at middle; with vertical row of four setae. Anal stylet twice length of width; with two minute setae at base of two slender apical setae. Ventral apical seta not much longer than anal stylet; dorsal seta twice length of stylet (Fig. 6). Bulga of spermatheca longer than wide; cribriform area extended slightly beyond margin of bulga. Hilla narrowed at juncture of bulga and enlarged towards apex; apex usually twice width of base. Bursa copulatrix long; base with "C" shaped thin sclerite merging into long and broad duct of bursa copulatrix. Duct of bursa copulatrix with small sclerotizations but no associated sclerites outside of duct. Duct of spermatheca thickened and crinkled at exit of bursa copulatrix, narrowing towards spermatheca (Fig. 7).

Arixenia esau Jordan, 1909 Figs 10-12
Note. Arixenia esau is a robustly built, highly mobile earwig capable of transporting many fleas for significant distances (Fig. 10). Numerous specimens of L. signata were observed clinging to the hairs of A. esau. Two such observations were photographed  with the fleas in situ (Figs 11-12). The flea (Fig. 12) is grasping setae with five of its six tarsal claws (arrows). It was physically difficult to dislodge the fleas from A. esau whose body and legs are somewhat densely covered with fine long setae (Figs 11-12).

Discussion
Lagaropsylla signata was previously known only from the type series. Although the host was listed as Ch. plicata, no information of how the bat was collected (mist net), or its habitat (cave, open field, hollow tree, human dwelling, etc.) was provided. Banjuwangi, Java is only a general locality, since modern Banjuwangi is a sprawling metropolis at the eastern extreme of Java along the Straights of Bali. In our study, a total of 72 L. signata were collected from the bodies of A. esau (7♂, 15♀) and from bat guano on the cave floor (15♂, 35♀). Cheiromeles torquatus was not found in Deer Cave in our study. Evidence of its historical presence was documented by Cranbrook (2010) in Niah Cave from archaeological records from the Pleistocene (40,000 years ago). Niah cave is approximately 115 km from Deer Cave. The finding of both L. signata and L. turba on A. esau implies that they have both adopted the same phoretic vehicle to access a bat host. Such evidence suggests that C. torquatus is present in Deer Cave and may serve as the principle host for L. signata but the definitive bat host for L. signata is unknown.
Because of the association of A. esau (Fig. 10) and Xeniaria jacobsoni (Burr, 1912) (two closely related earwigs) and their potential bat hosts, a discussion of the known biology of each follows. Nakata and Maa (1974) and Marshall (1977) provided assessments of the dermapteran suborder Arixeniina and their associated molossid bats. The phylogenetic position of A. esau as sister group to one lineage of free-living spongiphorid earwigs in the genus Marava Burr, 1911has recently been proposed (Naegle, et al. 2016. Medway (1958) documented colonies of C. torquatus in a "cave at Niah" (Subis Cave) in Sarawak, approximately 115 km from Deer Cave. From Subis Cave, Medway collected 23 fleas from a juvenile (non-flying) C. torquatus that had fallen from the roost 250 feet above the chamber floor. He noted that fleas were also widespread on the floor and were often attached to living Arixenia. These fleas, plus two additional males of L. turba that were collected in a hollow tree in Ulu Gombak, Selangor, Malaysia, comprised the type series from which Smit described L. turba. A bat was not associated with the Ulu Gombak collection. Arixenia esau was documented on the bodies of C. torquatus by Jordan (1909) and Medway (1958), while Cloudsley- Thompson (1957) reported "17 specimens of A. esau from bats harvested from a durian (Durio sp.) tree cavity" in addition to 168 specimens roaming free within the same tree cavity. Nineteen specimens of X. jacobsoni were also reported by Cloudsley-Thompson (1957) from the same durian tree cavity, but none were collected from bats. Medway and Chong (1969) documented A. esau on four mist-netted specimens of Ch. plicata in Pulai, Kelantan, and Borneo. Four specimens of X. jacobsoni were also reported by Burr (1912) from a cave near the shore of Babakan, Banjoumas Residency, Java. Further historic records of X. jacobsoni documented by Cloudsley-Thompson (1957) included two or three specimens from Mops mops (de Blainville, 1840) in Kuala Lumpur, Malaysia in July 1919, seven specimens from M. mops in "Malaya" in 1920, an unspecified number from Mindanao, Philippines, and 35 specimens from South Java (Banjoemos) from cave near Babakan in 1933. The presence of two earwig species occurring in the same cave environments and both documented on different bat species remains an enigma. It appears that A. esau is associated only with C. torquatus, while X. jacobsoni has been found on M. mops, Ch. plicata, and C. torquatus. Xeniaria jacobsoni has been collected on only one occasion on the body of C. torquatus but is found together with A. esau in common environs. The discovery of multiple specimens of L. signata attached to A. esau (behavior similar to L. turba) with no evidence of L. signata on any bat species will require further identification of bats in Deer Cave (and other caves) that may prove to harbor L. signata. The known distribution of bat hosts of Arixeniina species and the geographic distribution of Lagaropsylla spp. known from these areas are summarized in Table 1. Arixenia esau and X. jacobsoni have been associated in the hollows of trees but not in caves, while Xeniaria bicornis Maa, 1974 andArixenia camura Maa, 1974 have been found together in the hollows of trees but neither have been found in caves (Nakata and Maa, 1974). The latter two species found only in Mindanao were reported by Nakata and Maa (1974) from Ch. plicata and C. torquatus, respectively. Note: Cheiromeles torquatus does not occur in Mindanao, although Cheiromeles parvidens, a close relative, does. Arixenia camura, identified by Nakata and Maa (1974) would better be referred to C. parvidens and not C. torquatus. Medway (1958) observed negative geotropic behavior of A. esau, many falling from associated roosting bats at heights 250 feet above, and that their principle foods were insects and glandular skin of C. torquatus. Medway made no mention of earwigs on the cave walls. Burr and Jordan (1913) observed X. jacobsoni in countless numbers on the surface of guano and everywhere on the rocky walls at Gouwa Lawa (bat-cave), at Babakan, Java. This would suggest that X. jacobsoni (and likely A. esau as well) could serve as the principle vehicle for fleas (developing in the guano on the cave floor, or in tree hollows) to ride to the roosting bats located high on the ceilings of huge caves. It is highly unlikely that adult fleas would be capable of making the journey from the cave floor to the bat hosts hundreds of feet overhead. In the massive caves that harbor C. torquatus, these bats never make contact with the cave floor unless they fall to the floor as juveniles (which is fatal to the pups), or adults that bump into walls and are temporarily stunned. The inappropriate behavior of bumping into things (attributed to the presence of artificial light) was observed by Kirk-Spriggs (1989). Both earwig species feed on insects but X. jacobsoni feeds more voraciously on insects than does A. esau, as their diet also includes cannibalism of their own kind, especially those that are vulnerable during molting (Burr and Jordan 1913). Popham (1962) studied the morphology of the mouthparts of both species and concluded that the mouthparts of A. esau are more specialized for feeding (grazing) on the peculiar hairless, glandular exudates on the skin of C. torquatus, whereas the mouthparts of X. jacobsoni are less specialized and more suited to feeding on insects (although it has been found on M. mops, Ch. plicata, and C. torquatus). The differences in the recorded occurrence of A. esau and X. jacobsoni on different bat species may be a reflection of their differences in mouthpart morphology, dictating an ability to feed on the different skin (and hair) types. Jordan (1909) made reference to A. esau occurring in the sack formed by the membrane of the wings of C. torquatus. Not to be confused with the gular pouch, Kitchener (1954) described the function of these pouches. With the wings inserted into these pouches (by manipulations of the hind legs and feet), they are neatly folded around the bat, facilitating a quadrupedal mode of mobility within their roost areas (caves, crevasses, and hollow trees). Schutt and Simmons (2001) also addressed the function of the sub-axillary pouches and their relation to quadrupedal mobility. Arixenia esau might have a lesser chance of falling from the highly active aerobatics of their bat hosts if they seek these protected areas during bat flight. According to Medway (1958), the gular pouch is not sufficiently large enough to accommodate Arixenia.
Cheiromeles torquatus is distributed in southern peninsular Thailand, Malaysia, the insular portions of Indonesia (Java, Sumatra, Borneo), and Palawan, Philippines (Schutt and Simmons, 2001). The distribution of A. esau likely follows that of this bat host (although the distribution of A. esau is less well defined than its host). Although C. torquatus is listed in the IUCN Red List of Threatened Species, version 3.1, as "Least Concern", studies and assessments are particularly urgent because it is rare in some areas of its range and destruction of many habitats (hollow trees in forested areas) are being destroyed along with logging and human encroachment. The distribution of X. jacobsoni appears to be much broader, but may be a reflection of greater collecting activities.
Both L. turba and L. signata appear to require earwigs to transport them to a viable bat host that would otherwise be inaccessible. Such obligate phoretic behaviors requires additional studies, especially to elucidate the association of L. signata and its yet unknown bat host species.


extfieldsofstudy: Chemistry Medicine

text:
Prevention of Remifentanil Induced Postoperative Hyperalgesia by Dexmedetomidine via Regulating the Trafficking and Function of Spinal NMDA Receptors as well as PKC and CaMKII Level In Vivo and In Vitro

Remifentanil-induced secondary hyperalgesia has been demonstrated in both animal experiments and clinical trials. Enhancement of N-methyl-D-aspartate (NMDA) receptor trafficking as well as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) have been reported to be involved in the induction and maintenance of central sensitization. In the current study, it was demonstrated that dexmedetomidine could prevent remifentanil-induced hyperalgesia (RIH) via regulating spinal NMDAR-PKC-Ca2+/ CaMKII pathway in vivo and in vitro. We firstly investigated the effect of dexmedetomidine, a highly selective α2-adrenergic receptor agonist, on mechanical and thermal hyperalgesia using a rat model of RIH. NMDA receptor subunits (NR1, NR2A and NR2B) expression and membrane trafficking as well as PKC and CaMKII expression in spinal cord L4–L5 segments were measured by Western blot analysis. The expression of NMDA receptor subunits (NR1, NR2A and NR2B) were also detected by immunohistochemistry. Further more, the effect of dexmedetomidine on NMDA receptor current amplitude and frequency in spinal cord slices were investigated by whole-cell patch-clamp recording. We found that remifentail infusion at 1.2 μg.kg−1.min−1 for 90 min caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Subcutaneously injection of dexmedetomidine at the dose of 50 μg/kg at 30 min before plantar incision significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was dose-dependently attenuated by dexmedetomidine. These results suggest that dexmedetomidine can significantly ameliorate RIH via modulating the expression, membrane trafficking and function of NMDA receptors as well as PKC and CaMKII level in spinal dorsal horn, which present useful insights into the mechanistic action of dexmedetomidine as a potential anti-hyperalgesic agents for treating RIH.

Introduction
As potent analgesics, opioids are available for relieving moderate to severe pain.
Meanwhile, their prolonged usage may paradoxically be related to the development of opioid-induced hyperalgesia (OIH) [1][2][3]. OIH is characterized by decreased pain threshold and increased sensitivity to noxious stimuli [2,3]. Remifentanil is a μ-opioid receptor agonist for clinical anesthesia, however, it was claimed that remifentanil infusion might lead to OIH more frequently than other opioids because of having a rapid onset and short half-time of action [4].
Although the mechanisms underlying OIH have not been clearly illuminated, a certain amount of experiments suggest that it is associated with N-methyl-D-aspartate receptor (NMDAR)-protein kinase C(PKC)-Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) pathway [5][6][7][8]. NMDAR, an ionotropic glutamate receptors, is a protein complex composed of three classes of subunits: the essential subunit NR1, the modulating subunit NR2 (A-D), NR3 (A, B). Membrane trafficking of NR1 and NR2B subunit has been observed in incisional pain rat model after remifentanil infusion through glycogen synthase kinase-3β (GSK-3β) pathway [9,10]. PKC is a family of serine/threonine kinases distributed within the CNS, which consists of at least 12 isoforms [5]. Calcium dependent PKCγ isoform identified in lamina II of superficial spinal dorsal horn, an area implicated in pain, is considered to be a critical regulator of central sensitization by alleviating Mg 2+ block of NMDA currents [6]. Ca 2+ / CaMKII is a multifunctional serine/threonine protein kinase co-localized with the μ-opioid receptor in superficial spinal dorsal horn [7,8]. It is implicated that CaMKIIα is a critical cellular signaling mechanism leading to and maintaining OIH [8].
Dexmedetomidine, a highly selective α2-adrenergic receptor (α2AR) agonist, possesses sedative, anxiolytic, analgesic, and hemodynamic-stabilizing properties [11,12] without significant respiratory depression [13]. Its greater affinity to α2AR and shorter duration of action have led to its usage as an adjuvant for patients undergoing mechanical ventilation in general anesthesia [2,14]. It has been demonstrated that systemic administration of dexmedetomidine dramatically enhances analgesic effect of opioids and reduces perioperative analgesic requirements [11,12,15]. It has been suggested that antihyperalgesic effect of dexmedetomidine is linked to depression in spinal NMDAR-mediated synaptic transmission and dexmedetomidine may be an option to improve pain control for OIH patients [16,17]. However, whether NMDAR-PKC-CaMKII pathway served as a target for preventing OIH was still needed to discuss.
The present study was conducted to investigate the efficacy of dexmedetomidine on remifentanil-induced postoperative hyperalgesia in an incisional pain rat model. We also wonder whether the antihyperalgesic effects of dexmedetomidine is associated with membrane trafficking of NMDAR and expression of PKC and CaMKII. In order to verify the effects, remifentanil was administrated intravenously and dexmedetomidine was administrated subcutaneously. The whole cell patch clamp was also applied to prove that whether miniature excitatory postsynaptic current (mEPSC) is relieved by dexmedetomidine via NMDA receptor pathway after management with remifentanil.

Ethics statements
All experimental procedures and protocols were approved by the Institutional Animal Care Committee of Tianjin Medical University and performed according to the "Policies on the Use of Animal and Humans in Neuroscience Research". The protocol was approved by the Committee on the Ethics of Animal Experiments of Tianjin Medical University General Hospital, Tianjin, China (Permit Number: 2015-X6-17). All surgery was performed under sevoflurane anesthesia, and all efforts were made to minimize suffering and to use the minimum number of animals necessary to obtain valid results.

Animals
Both adult (weighing 240-260 g) and newborn (14-21 days old) male Sprague-Dawley rats, obtained from the Laboratory Animal Center of the Military Medical Sciences Academy of the Chinese People's Liberation Army, were used in the experiment and housed with a 12 h light/ 12 h dark cycle. The rats were given free access to water and food ad libitum and kept in cages at a room temperature (18~22˚C) and humidity (40%~60%) in a clean animal house at least 3 days before drug treatment or surgery. The experiment protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University and was performed on the basis of the National Institutes of Health Guide for Care and Use of Laboratory Animals. No efforts were spared to minimize animal sufferings.

Experimental protocol
The rats were anesthetized under sevoflurane anesthesia (induction, 3.0%; maintenance, 1.0%; batch number: 100628; Maruishi Pharmaceutical Co., Osaka, Japan) via a nose mask. Then the rats were placed in plastic tubular restrainers. The middle and lower third of the tail was chosen as the puncture point. A 24-gauge venous indwelling needle filled with heparinized saline was inserted into the caudal vein.

Plantar incision
The incisional pain rat model was constructed as described previously [18]. A 1-cm longitudinal incision was made through the skin, fascia, and muscle in sequence of the left hindpaw after disinfection, starting 0.5 cm from the proximal edge under sevoflurane anesthesia (induction, 3.0%; maintenance, 1.0%) via a nose mask. Pay attention to divide the underlying flexor muscle but keep the muscles origins and terminations and attachment integrity. The skin was sutured without inverted, overlap, cracked by 4-0 silk sutures after compression hemostasis. Finally, erythromycin ointment was smeared to avoid infection. Postoperative monitoring of temperature, heart rate and condition of the condition (bleeding, pyosis and so on) should be done every 2 h. If the incision of rat was suppurative or bleeding all the time, some analgesics should be given and the rat was excluded.

Behavioral testing
Paw withdrawal threshold (PWT) was recorded by electronic von Frey filaments (BSEVF3, Harvard Apparatus, Edenbridge, UK) to measure mechanical hyperalgesia. After adult rats were placed individually in mental cages (20 cm×20 cm×15 cm) with a wire grid bottom for 10 min, Von Frey filaments were exerted vertically to the plantar side of the right hindpaw. Lifting of the hindpaw off the cage surface or flinching was regarded as a positive response. A critical value of 60 g was used to prevent paw damage, and each trial was repeated 3 times at 10-min intervals. The calculated average value was PWT.
Paw withdrawal latency (PWL) was recorded by a 55˚C hotplate (YLS-6B, Zheng Hua Biological Instrument Co., Anhui, China) to assess thermal hyperalgesia. A critical value of 60 seconds was used to prevent paw damage, and each trial was repeated 3 times at 10-min intervals. The calculated average value was PWL.

Western blot
After completing the last behavioral tests, rats were sacrificed under sevoflurane anesthesia. The chest was cut along the midline to expose the heart, then an infusion needle was inserted into the apical portion of the aorta, the right atrial appendage was cut and normal saline was infused rapidly until transparent liquid flowed out. Then a longitudinal incision along the dorsal midline was made and L4-L5 spinal cord segments were extracted rapidly and kept in liquid nitrogen. The segments were homogenized in ice-cold sodium dodecyl sulfate lysis buffer containing protease inhibitors (Sigma-Aldrich Co., St. Louis, MO, USA). Then the lysate was centrifuged at 14,000r for 10 min at 4˚C, and the supernatant was removed as total protein. A membrane compartment protein extraction kit (Biochain Institute, Inc., Hayward, CA, USA) was applied to extract the membrane protein. Eventually after the total protein was boiled at 95˚C for 10min and membrane protein was boiled at 65˚C for 10min, they were store at -20˚C. Samples were separated on 8%-10% SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in TBST for 2 hour and subsequently incubated overnight at 4˚C with mouse anti-rat epidermal growth factor receptor (EGFR, 1:1,000; MBL, Naka-ku Nagoya, Japan), monoclonal mouse anti-β-actin antibody (1:3000; Liangsen Biological Technology Co., Ltd, Shanghai USA), polyclonal rabbit antibodies against rat NR1 (

Immunohistochemistry
The L4-L5 spinal cord segments were extracted rapidly after the last behavioral test. 4% paraformaldehyde in phosphate buffered saline (PBS) was applied to fix the samples for 6 h, and then paraffin was used to embed the samples. The samples were cut into 7-μm-thick sections and placed onto glass slides. After deparaffinization and rehydration in descending concentrations of ethanol, the sections were treated with avidin-biotin peroxidase for 15 min. To make the results more intuitive, citrate solution was used for antigen retrieval. 5% normal goat serum was selected for treatment to block non-specific reactions for 1h at room temperature. Then the sections were incubated with primary antibody of rabbit polyclonal anti-NR1, NR2A, NR2B (1:200; Abcam, Cambridge, UK) overnight at 4˚C respectively. After rewarming for 45min, the sections were incubated with biotinylated secondary antibody (1:300, Boster Biological Technology, Ltd., Wuhan, China) for 30 min at room temperature and stained by diaminobenzidine (DAB substrate kit, Boster Biological Technology). Sections were counterstained with hematoxylin, differentiated by ethanol hydrochloride, dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with neutral gum. Images were obtained from an Olympus eclipse 80i microscope (Olympus, Tokyo, Japan).

Spinal cord slice preparation and whole-cell patch-clamp recording
Rat spinal cord slices were prepared as described previously. The postnatal (14-21 days old) rats were anesthetized with sevoflurane (induction, 3.0%; maintenance, 1.0%). The L4-L5 spinal cord segments were separated rapidly with the same method preciously, sliced into transverse slices (400 μm) with a vibratome (VT1000S, Leica, Germany), then incubated in ACSF at room temperature (22˚C-25˚C) and aerated with 95% O 2 and 5% CO 2 at pH 7.4 for 1 h. ASCF is composed of (in mM): 126 NaCl, 3.5 KCl, 2 MgCl 2 , 2 CaCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 and 10 D-glucose. The slices were then transferred to a recording chamber in succession. The chamber was perfused with oxygenated ACSF continuously and placed on an upright microscope (BX51W1, Olympus, Tokyo, Japan) and continuously perfused with oxygenated ACSF. Individual neurons were identified by a television monitor connected to the low light-sensitive CCD camera (710M, DVC, USA). Borosilicate glass microelectrodes with tip openings of 1-2 μm and electric resistance of 3-5 MO were drawn by vertical electrode puller (PIP5, HEKA, Germany) and applied to whole-cell patch-clamp recording. Microelectrodes were filled with an intracellular solution composing of (in mM) 130 KCl, 10 HEPES, 0.5 CaCl 2 , 10 EGTA, 2 MgCl 2 , 2 Mg-ATP and 0.3 Na-GTP at pH 7.3. CNQX (20 μM), tetrodotoxin (TTX 10 μM) and bicuculline (BIM 20 μM) were added into perfusion solution before recording to accurately verify the NMDAR miniature excitatory postsynaptic currents (mEPSC). In previous trials, AP-5 (a NMDA receptor antagonist, 2μM) was added after NMDA receptor-mEPSC was detected, and mEPSC fade away a few minutes later. Therefore, the mEPSC was mediated by NMDA receptors. ACSF without Mg 2+ is used to observe NMDA receptor-mediated synaptic transmission. All responses were recorded by an EPC 10 amplifier and Pulse 8.52 software (HEKA, Germany). Current data was filtered with an eight-pole, low-pass Bessel filter at 2.9 kHz and digitized at 10 kHz for off-line analysis. mEPSC was then analyzed by Clampfit 9.0 (Axon Instruments, USA). Total charge transfer by all mEPSC was determined for each condition in 5 min and repeated 8 times.

Statistical analysis
Statistical analysis was performed by SPSS 19.0 (IBM, Chicago, IL). All data were expressed as mean±standard deviation(SD). Time course data for both the thermal and mechanical hyperalgesia were analyzed by two-way ANOVA with repeated measures to detect interactions between treatments. ANOVAs with statistically significant interactions between treatments (P < 0.05) were followed by post hoc comparisons using Bonferroni's t test when appropriate. The results of Western blot, immunohistochemistry, amplitude and interevent intervals of NMDAR-mediated mEPSC in different groups, one-way ANOVA followed by Tukey HSD test were performed. Statistical analysis was performed with GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA). A P value<0.05 was considered statistically significant.

Remifentanil-induced postoperative mechanical and thermal hyperalgesia
There was no significant difference in the baseline PWT to mechanical stimuli and PWL to thermal stimuli among different groups (P >0.05). Compared with group C, PWT and PWL were significantly decreased at 2, 6, 24, 48 h in other groups except group D (P<0.01). Remifentanil infusion at the rate of 1.2 μg.kg −1 .min −1 for 90 min or plantar incision significantly decreased PWT and PWL at 2, 6, 24 and 48 h after plantar incision (P < 0.01, S1 Fig). Compared with group I and group R, intraoperative administration of remifentanil during incision operation significantly decreased PWT and PWL (P < 0.01, S1 Fig). Dexmedetomidine administration individually had no effect on PWT and PWL compared with group C. However, dexmedetomidine administration before plantar incision induced a remarkable increase in PWT and PWL at 2, 6, 24, 48 h compared with group R+I (P <0.01, S1 Fig). Remifentanil-induced postoperative hyperalgesia increased the expression of NR1 and NR2B subunit in the plasma membrane, but not NR2A subunit in the spinal cord Compared with group C, total (t) and membrane (m) expression of NR1, NR2B and the ratio of m/tNR1, m/tNR2B was significantly increased after incision or remifentanil infusion (P < 0.01, S2A, S2C and S2E Fig). The combination of surgery and remifentanil infusion could magnify the enhancement of the membrane and total expression of NR1 and NR2B subunits induced by incision or remifentanil respectively (P < 0.01, S2A, S2C and S2E Fig). Dexmedetomidine administration individually had no effect on NMDAR trafficking compared with group C. However, dexmedetomidine administration before plantar incision caused a significant reduction in the expression of mNR1, tNR1, mNR2B, tNR2B and the ratio of m/tNR1, m/tNR2B compared with group R+I (P <0.01, S2 Fig). No considerable changes were observed in mNR2A, tNR2A and the ratio of m/tNR2A (P > 0.05, S2C and S2D Fig).
The above data showed that remifentanil infusion and plantar incision might induce hyperalgesia by increasing membrane trafficking of NR1 and NR2B subunit, while dexmedetomidine might produce a preventive effect via inhibiting the trafficking process. The changes of NR1, NR2B expression detected by immunohistochemistry staining in dorsal horn were the same to that of Western blot (P< 0.05, S3B, S3C, S3D, S4B, S4C and S4D Figs), while the expression of NR2A was still the same (P>0.05, S3C and S4C Figs).

Dexmedetomidine decreases remifentanil-induced NMDAR function enhancement in dorsal horn neurons
To further confirm the effect of dexmedetomidine on NMDAR -mediated mEPSC in spinal dorsal horn neurons, we examined NMDAR-mediated mEPSC with co-incubation of 4 nM remifentanil and 2 nM, 4 nM and 6 nM dexmedetomidine. Both frequency and amplitude of NMDAR-mediated mEPSC were dose-dependent decreased by dexmedetomidine compared with group R (P < 0.01, S6A, S6B and S6C Fig). The above data suggested that dexmedetomidine could attenuate NMDAR function enhanced by remifentanil in dorsal horn neurons.
The current study showed that remifentanil infusion and incision could induce thermal and mechanical hyperalgesia in a rat model of incisional pain, which was prevented by dexmedetomidine. The enhancement of membrane and total NR1 and NR2B subunits expression together with PKCγ, CaMKIIα and pCaMKIIα expression were observed in spinal dorsal horn after remifentanil infusion and incision, but there was no change in NR2A subunit. The expression was also decreased by dexmedetomidine. In whole-cell patch-clamp experiment, the amplitude and frequency of NMDAR-mediated mEPSC were enhanced by 4 nM remifentanil infusion but dose-dependent attenuated by dexmedetomidine. The above data suggested that α2AR agonist dexmedetomidine could depress remifentanil-induced hyperalgesia via regulating subunits trafficking and function of NMDAR as well as PKCγ, CaMKIIα and pCaM-KIIα expression in spinal dorsal horn.
The rat model of incisional pain was performed in accordance with Brennan described to simulate remifentanil-induced postoperative hyperalgesia [18]. The reason why sevoflurane was chosen as the anesthetic through the entire experiment was because it had no impact on behavioral tests [9]. The dosage of remifentanil was a key factor in present study. Remifentanil infusion at a rate of 1.2 μg.kg −1 .min −1 could evoke hyperalgesia in rats, and continuous infusion for 60~90 min led to significantly reduction of mechanical pain threshold [9,10,19]. Therefore, remifentanil was infused at a rate of 1.2 μg.kg −1 .min −1 for 90 min in this study. It was tried out that PWT of tissues around the incision was prolonged than that of incisional group after 50 μg/kg dexmedetomidine was injected subcutaneously. Several studies demonstrated that hyperalgesia induced by remifentanil occurred at 2 h and reached the peak at 24-48 h after administration [10,20]. Combination of remifentanil infusion and incision could cause greater PWT and PWL compared with remifentanil infusion or incision alone in our previous studies [9,10,19]. Our behavioral results suggested that either remifentanil infusion or surgery alone induced hyperalgesia to mechanical and thermal stimuli, which was greater when remifentanil administration and surgery are combined. To conduct a better research about the role of dexmedetomidine, we set remifentanil infusion and incision group as the pain group. We did the experiment about dexmedetomidine and results suggested that dexmedetomidine did not change PWT and PWL at the dose of 50 μg/kg. It suggested that the dose of dexmedetomidine had no effect on pain process.
The whole cell patch clamp was used to detect NMDAR-mediated mEPSC of neurons in spinal dorsal horn to distinguish presynaptic and postsynaptic response. On the basis of synaptic vesicular release quantum theory, miniature postsynaptic currents are supposed to represent the spontaneous release of neurotransmitter or vesicles from presynaptic membrane. The frequency of NMDAR-induced mEPSC is considered as the presynaptic effects, whereas the amplitude is thought to reflect postsynaptic effects [21,22]. Therefore, the function of NMDAR was evaluated by the frequency and amplitude of NMDAR-mediated mEPSC [21]. 4 nM remifentanil for 60 min increased both frequency and amplitude of NMDAR-mediated mEPSC significantly in previous study. We did the experiment about dexmedetomidine and results suggested that dexmedetomidine alone did not change NMDAR-mediated mEPSC. However, both frequency and amplitude of NMDAR-mediated mEPSC enhanced by remifentanil were dose-dependent decreased by dexmedetomidine. The above data suggested that dexmedetomidine could attenuate NMDAR function enhanced by remifentanil in dorsal horn neurons. The results that up-regulation of NR1 and NR2B subunits membrane trafficking was consistent with function of mEPSCs recorded.
Numerous studies were conducted to explore the mechanism of OIH, but there were still different viewpoints on it. The majority of academics suggest that it is associated with acute receptor desensitization caused by G-protein receptor decoupling, upregulation of cAMP pathway and NMDAR activation [23]. NMDAR activation accounts for OIH and tolerance processes, which is in accord with that NMDAR antagonist ketamine was able to inhibit central sensitization stated in experimental studies performed in both animals and volunteers [24]. So NMDAR has recently been considered to play a crucial role in synaptic plasticity and chronic pain formation [24]. The NR2 subunits amplify NR1 activity and these subunits coassemble in various combinations to induce functional variability in NMDAR signaling [25]. The up-regulation of expression and membrane trafficking of NR1 and NR2B subunits in spinal cord after remifentanil administration and incision verified in the experiment was in consistent with our previous results [9,10]. The results showed that remifentanil infusion and plantar incision might induce hyperalgesia by increasing the expression and membrane trafficking of NR1 and NR2B subunit detected by western blot and immunohistochemistry staining. It is worth noting that NR2B subunit plays a dominant role in sensory pathway of spinal dorsal horn. Moreover, there is a substantial amount of evidence that tyrosine phosphorylation of NR2B on distal C-terminus at Tyr1472 in spinal dorsal horn may contribute to NMDAR activation and development of OIH [20,26].
It has been proposed that Ca 2+ influx via activation of NMDAR results in PKCγ and CaM-KIIα activation as well as CaMKIIα autophosphorylation at position Thr286, leading to NMDAR phosphorylation and activation and thus Ca 2+ influx through the channels [8,25,27,28]. The positive feedback would potentiate PKCγ and CaMKIIα activity and intracellular Ca 2+ level, leading to neuronal excitability [29]. Furthermore, PKCγ indirectly potentiates NMDAR hypersensitization by activating a downstream signalling peptide of the tyrosine kinase (Src) signalling cascade [27,30,31]. Thus, translocation of PKC from cytoplasm to cell membrane is a sensitive indicator of activation [32]. Increasing evidence indicates that the distribution and function of NMDAR is modulated by PKC via regluating the interactions between NMDAR, postsynaptic density (PSD) and cytoskeletal proteins [33]. PKC activation increases substance P as well [34]. S1303 and S1323 serine residues in C-terminal region of the NR2B subunit are directly phosphorylated by PKC and enhance the PKC-mediated NR2B/ NR1 currents [27,30]. In addition, S1303 had been confirmed as a major site of CaMKII phosphorylation [27]. Neurogranin (Ng), previously known as RC3 or P17, is a postsynaptic neuron specific protein that serve as a link between PKC and CaMKII [35]. A large increase in intracellular Ca 2+ prompts the release of CaM by dissociating CaM/Ng complex. Latter in the presence of Ca 2+ , Ca 2+ /CaM-dependent enzymes such as CaMKII is activated [36]. In contrast, low Ca 2+ level favors CaM/Ng formation and activates CaMKII [37]. Alternatively, CaM/Ng complex may also be dissociated via Ng phosphorylation by PKC [36,38]. OIH was absent in mice lacking gene coding for PKCγ and CaMKIIα T286A mutant mice [39]. Pretreatment with the PKCγ agonist resulted in CaM release, pCaMKII formation and development for OIH.
The α2 adrenergic receptor agonist dexmedetomidine may act on spinal α2 receptor. α2 adrenoceptor is also G-protein coupled receptors. They exert their effects through inhibition of cAMP formation and ultimately reduction in PKA activity once activated. Spinal PKA activation enhanced the activity of Fyn kinase that is major tyrosine kinase phosphorylates NR2B at Tyr1472, and then potentiate NMDARs functions [26,40]. Anti-hyperalgesic effect of dexmedetomidine may depend on its ability to modulate spinal NMDAR activation by suppression of NR2B phosphorylation via a Fyn-dependent mechanism. It was in consistent with our previous results that dexmedetomidine might produce a preventive effect via inhibiting the NR1 and NR2B subunits trafficking process. And thus reduction in Ca 2+ influx resulted in decreased PKC activation, CaMKIIα activation and autophosphorylation. In addition, several studies demonstrated that dexmedetomidine had a high affinity for I 2 imidazole receptors. Both α2 adrenergic receptor and I 2 imidazole receptors could provide analgesic effects in spinal cord.

Conclusions
In conclusion, the present study indicates that antihyperalgesic effects of dexmedetomidine may be associated with enhanced membrane trafficking and expression of NMDAR, expression of PKC and CaMKII and current in both presynaptic and postsynaptic levels. Dexmedetomidine may be a potential new drug target to treat remifentanil-induced hyperalgesia. bicuculline (BIM, 20 μM) and AMPAR antagonist CNQX (20 μM), NMDAR-mediated mEPSCs were recorded at the holding potential of −70 mV. Representative traces of mEPSCs under blank control (group C), remifentainil 4 nM(group R), dexmedetomine 4 nM(group D), dexmedetomine 2 nM + remifentainil 4 nM(group D1), dexmedetomine 4nM + remifentainil 4 nM(group D2), dexmedetomine 6 nM + remifentainil 4 nM(group D3) showed in graph A. Scale bar, 100 pA, 30 s. The Bar chart of mEPSCs frequency and mEPSCs amplitude were showed in graph B and C. Data were expressed as mean±SD. Compared with the C group, Ã P < 0.01; compared with the R group, # P < 0.01; compared with the D1 group, $ P < 0.01; compared with the D2 group, & P < 0.01; compared with the D3 group, § P < 0.01; N = 8, analysis of variance. (TIF)


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PICS bags safely store unshelled and shelled groundnuts in Niger

We conducted an experiment in Niger to evaluate the performance of hermetic triple layer (Purdue Improved Crop Storage- PICS) bags for the preservation of shelled and unshelled groundnut Arachis hypogaea L. Naturally-infested groundnut was stored in PICS bags and woven bags for 6.7 months. After storage, the average oxygen level in the PICS bags fell from 21% to 18% (v/v) and 21%–15% (v/v) for unshelled and shelled groundnut, respectively. Identified pests present in the stored groundnuts were Tribolium castaneum (Herbst), Corcyra cephalonica (Stainton) and Cryptolestes ferrugineus (Stephens). After 6.7 months of storage, in the woven bag, there was a large increase in the pest population accompanied by a weight loss of 8.2% for unshelled groundnuts and 28.7% for shelled groundnut. In PICS bags for both shelled and unshelled groundnuts, by contrast, the density of insect pests did not increase, there was no weight loss, and the germination rate was the same compared to that recorded at the beginning of the experiment. Storing shelled groundnuts in PICS bags is the most cost-effective way as it increases the quantity of grain stored.

Introduction
Groundnut (Arachis hypogaea L.) is one of the most important cash crops in West Africa. Groundnuts generates about 60 percent of farmers' income in the region, and as much as 80% in Senegal (Ntare et al., 2004;Sylla, 2010). West Africa produces about 5.87 million tons of groundnuts. This accounts for about 67% of Africa's total production and 16% of world production (Ntare et al., 2004). Groundnuts are grown in West Africa as a source of protein (between 24 and 35% depending on the variety) and carbohydrates (Rehm and Espig, 1991). They are also an important crop because of their high content in oil, which is in the range of 38e50% (Ribier and Rouzi ere, 1995). Frequently grown by smallholder farmers, groundnuts are often considered a woman's crop. In Niger, groundnut is the fourth most important crop. It is cultivated on more than 700,000 ha with an estimated 402,000 tons produced each year (M.A, 2015). In Niger, groundnut is used to produce cooking oil, and its paste is used to prepare local dishes.
In West Africa, groundnuts are stored in many types of containers including clay pots, jute and woven plastic bags, mud silos, plastic and metal drums, and glass jars (Masters et al., 2013). These containers provide little or no protection against insect pests. Different approaches are used to manage pests during groundnuts storage. Drying unshelled groundnut below 7% helps reduce insect infestations and incidence of aflatoxin. Natural or synthetic insecticides may help preserve groundnut quality during storage (Gu eye et al., 2011;Conway, 1975;Putnam et al., 1991). Insecticide treatment may be required for groundnuts destined for export (APEDA, 2015). Several chemicals are recommended for insect control including fumigants and, powdered and liquid insecticides (Ranga Rao et al., 2010).
Hermetic storage such as the Purdue Improved Crop Storage (PICS™) bags have shown to be effective in preserving many cereal and legume crops in Sub-Saharan Africa (Baributsa et al., 2010;Baoua et al., 2014a;Murdock et al., 2012). PICS bags serve both as storage containers and also as postharvest storage pest management tools. These bags have already been tested for the protection of groundnuts against C. serratus and aflatoxin contamination in India (Sudini et al., 2015). After 4 months of storage in small custom-made hermetic bags holding 2 kg, groundnut quality including germination was well maintained. No study has been conducted in West Africa on the effectiveness of hermetic PICS bags under Sahelian conditions. The purpose of the present study were: (i) to evaluate the performance of PICS bags for storing naturallyinfested shelled and unshelled groundnuts in Niger, and; (ii) identify the insect pests associated with stored groundnuts in Niger.

Materials and methods
An experiment was conducted at the Maradi Station of the National Institute of Agricultural Research e Niger (INRAN). Unshelled and shelled groundnuts were stored in 50 kg capacity PICS triple-layer bags (manufactured by Lela Agro Industries Ltd.; Kano, Nigeria) and in regular woven bags as the control. PICS bags consist of two high-density polyethylene (HDPE) liners of 80 mm wall thickness, fitted inside a woven polypropylene bag to enable handling. Naturally-infested shelled and unshelled groundnuts produced the previous season were purchased in a local market in Maradi. The experiment had two treatments: (1) PICS bags in four replicates and (2) woven bags in four replicates. Bags were filled with either 20 kg of unshelled groundnuts or with 10 kg of shelled groundnuts. Before filling the bags, each type of groundnut was physically and thoroughly mixed to ensure a uniform insect infestation. Replicate treatments were held on pallets in a storehouse under ambient local conditions. Although this does not allow control of temperature and humidity, it closely duplicates conditions under which groundnuts are stored in the real world of representative African farmers. Descriptions of how the PICS technology is used are available in several languages on the PICS website (https://picsnetwork.org/resources/?tab_id¼Posters).
The experiments were initiated on 8th April 2015 and terminated on 27th October 2015 after a period of 202 days or 6.7 months. The following data was collected at the beginning and the end of the experiments: (i) Numbers of insects and damage levels were determined by randomly selecting three separate samples of 250 g of groundnuts from each bag (shelled and unshelled). Each sample was sieved and shelled (for unshelled groundnuts) to separate and count live adults and larvae. Pupae were counted as adults. One hundred seeds were picked randomly from each sample, weighed and then the number of seeds bearing eggs and holes determined by counting. (ii) To determine weight loss, a random sample of 100 grains or 100 pods were weighed at the beginning and the end of the experiment. The percent weight loss was estimated using the following formula-percentage weight loss ¼ [(Initial weight -weight after 6.7 months)/(Initial weight)] *100. (iii) Germination was assessed as follows: (a) one hundred seeds from each sample were divided into four Petri dishes of 25 seeds each; the dishes were first lined with glass wool disinfected with 5% bleach; (b) distilled water was added to each dish to wet the wool; (c) germination was noted at up to 7 days. (iv) The oxygen (O 2 ) and carbon dioxide (CO 2 ) concentrations in each PICS bag were determined using a Mocon PAC Check ® Model 325 Headspace analyzer (Mocon, Minneapolis, MN). Measurements were made on each PICS bag before closing and at the end of the experiment using a hypodermic needle to penetrate into the interior of the bag though a small area from which the woven outer bag had been removed. (v) At the end of the experiments the two inner HDPE liners were examined to determine the number of holes made by insects and inspected for any other physical changes. (vi) In addition, we estimated the shelling percentage of the unshelled groundnuts by weight and by volume. Twelve samples of unshelled groundnuts of 200 g each and 12 samples of 250 ml each were used to estimate the shelling percentage for weigh and volume, respectively. The shelling percentage for weight was determined by dividing the weight after and before shelling x 100; while the shelling percentage for volume was calculated by dividing the volume after and before shelling x 100. The shelling percentage helped determine the efficiency of storing shelled compared with unshelled groundnuts in PICS bags.
Analysis of variance followed by the StudenteNewmaneKeuls (SNK) test was used to compare the means of infestation levels, insect damage and germination rate for groundnuts stored in PICS bags and woven bags (control) after 6.7 months of storage. The T test was used to compare the means O 2 and CO 2 levels in PICS bag at the beginning and the end of experiment. Statistical analysis was done with Statistical Package for the Social Sciences (SPSS) Version 16.0, IBM (Chicago, Illinois).

Results
Unshelled groundnuts: At the outset of the trial unshelled groundnuts had an average initial infestation of 7 living insects per sample of 250 g (Table 1). These were mainly adults of T. castaneum (50.74%), C. cephalonica larvae (40.29%) and C. ferrugineus adults (8.95%). After 6.7 months of storage, there was 100% mortality for each insect pest in PICS bags, while in the control woven bags the population of T. castaneum had increased 2.7-fold, C. cephalonica 6fold, and C. ferrugineus 3.2-fold. There was also a shift in the relative numbers of the three species after 6.7 months of storage. T. castaneum now made up 33.6% of the insect population, C. cephalonica 59.5% and C. ferrugineus 6.9%.
Shelled groundnuts: Shelled groundnuts had a mean initial infestation of 0.3 living insects per 250 g (Table 1). Of these, 66.7% were T. castaneum adults and 33.3% were larval C. cephalonica. At the end of the experiment, in the PICS bags, the densities of the pest were not different from those initially present. However, in woven bags the density of adults of T. castaneum was 174 times higher than the initial number while the density of C. cephalonica did not differ from that originally recorded. The proportions of the total insect pest population in the woven bags at the end of the experiment were 76.1% for T. castaneum, 0.44% for C. cephalonica and 23.5% for C. ferrugineus. Though not visibly evident at the outset of the experiment, after 6.7 months of storage C. ferrugineus averaged 11 adults per 250 g of shelled groundnut.
The weight of samples of shelled and unshelled groundnuts stored in PICS bags after 6.7 months of storage did not differ from that recorded at the beginning of the experiments (Table 1). By contrast, in woven bags we noted an average weight loss of 8.2% for unshelled groundnut and 28.7% for shelled groundnuts (Table 1). Unshelled groundnuts used in this experiment had an initial shelling percentage of 67.3± 1.6% in relation to the weight and 29.3± 4.6% in relation to the volume (data not presented). Germination rates at the end of the experiments for both unshelled and shelled groundnuts stored in PICS bags were comparable to those observed at the beginning of the trial (Table 2). By contrast, both types of groundnuts stored in regular woven bags had reductions in germination amounting to 14.9% for unshelled groundnuts and 64.8% for shelled groundnuts ( Table 2).
The initial mean oxygen levels of about 21% (v/v) decreased by 2% for unshelled and 5% for shelled groundnuts stored in PICS bags for 6.7 months (Table 3). Carbon dioxide was below the level of detection at the beginning of the experiment and increased only a little after 6.7 months of groundnut storage in PICS bags. At the end of the experiment, all PICS bags were inspected and no alterations or perforations were observed on any outer or inner HDPE liners (data not shown).

Discussion
Our results establish that PICS bags are effective at suppressing storage pests and maintaining grain quality during shelled and unshelled groundnut storage in the West African nation of Niger. T. castaneum and C. cephalonica species were the most important pests we found in naturally-infested groundnut stored for about 7 months. They are well-known pests of groundnut that affect food quality and seed viability (Nautiyal, 2002;Ranga Rao et al., 2010;Delobel and Matokot, 1990). After nearly 7 months of storage in PICS bags, pest population growth was suppressed in both shelled and unshelled groundnuts. When infested grain was stored for 6.7 months in woven bags, the population of C. cephalonica was suppressed in favor of T. casteneum in shelled groundnuts. C. ferrugineus was a minor pest at the beginning of the experiment in unshelled groundnuts and was not found in shelled groundnuts. C. ferrugineus is considered a secondary pest on groundnuts and is often found in association with other pests (Nautiyal, 2002;Sallam, 2008;Raoul and L eonard, 2013). It was observed at the end of the experiment that C. ferrugineus developed well when T. castaneum was thriving on shelled groundnut stored in woven bags.
Groundnuts (shelled and unshelled) stored in PICS bags experienced minimal weight loss, less than 1%. Shelled groundnuts stored in woven bags were damaged mainly by T. castaneum while the unshelled groundnuts was mainly damaged by C. cephalonica. While unshelled groundnuts had an initial higher insect infestation level (23 times) than shelled groundnuts, their weight loss was less affected at the end of the experiment. Weight loss of unshelled and shelled groundnuts kept in woven bags was 8.2% and 28.7%, respectively. Nautiyal (2002) reported that T. castaneum is a major pest of shelled groundnuts and can causes weight losses of 4.5%. In our experiments in Niger T. castaneum caused substantial damage to shelled groundnuts, more than 7 times that reported by Nautiyal (2002). The damage resulting from C. cephalonica is well documented for other crops. For example, with maize in India it leads to 7.46% weight loss after 2 months of storage (Meena et al., 2014). With cocoa beans in Ghana it causes weight loss of 13.34% after 4 months of storage (Azalekor, 1999). As is well known, the groundnut shell helps protect the seed from damage by storage insects. Some producer organizations recommend keeping groundnuts in the shell form when storage is for a period exceeding 6 months (http://www.rongead.org/IMG/pdf/BAI_stockage_ arachide.pdf). C. serratus, despite its reputation for causing Table 1 Insect infestation and damage to shelled and unshelled groundnut kept in 50 kg Purdue Improved Crop Storage (PICS) bags and woven bags filled with naturally infested grain (by Tribolium castaneum, Corcyra cephalonica and Cryptolestes ferrugineus) and stored for 6.7 months. Means followed by the same letter are not significantly different (LSD, 5%).
Groundnut types Treatments n Germination rate (%) Unshelled Initial 48 83.7 ± 1.6 a PICS bag after 6.7 months 24 81.1 ± 1.6 a Woven bag after 6.7 months 24 68.8 ± 2.1 b Shelled initial 48 87.8 ± 1.5 a PICS bag after 6.7 months 24 88.8 ± 2.6 a Woven bag after 6.7 months 24 23.0 ± 3.9 b damage to stored groundnuts, was not observed in our experiment in Niger. However, several authors have reported its presence in the Sahelian zone (Aitken, 1975;Gu eye et al., 2011). While storage in unshelled form helps farmers deal with insects, storing unshelled groundnuts uses substantially more volume in the container or stores. Storing groundnuts in shell increases the cost of storage by reducing the quantity of grain stored by a third. This can involve additional costs for buying bags and storage space. Therefore, the use of the PICS bag for shelled groundnut appears to be the most effective and economical way of storing groundnuts. Oxygen levels in PICS bags decreased for both shelled and unshelled groundnuts after 6.7 months of storage, with the decrease more pronounced in shelled groundnuts. The levels of oxygen in this study were similar to those observed by Sudini et al. (2015) in an experiment conducted on groundnut pods in India but less than has been observed with other stored grains. The decrease in oxygen may explain the cessation of pest development. Since O 2 and CO 2 levels were not measured except at the start and end of the experiment, it is possible that O2 levels fell well below the final value observed and then, after the insect population had ceased activity or died, slow leakage of O2 back into the bag may have occurred. Further studies are needed to determine if this in fact occurred. It is well known that the conditions of hypoxia and hypercarbia disturb insect metabolism and can result in death (Hochachka, 1986;Weyel and Wegener, 1996;Ofuya and Reichmuth, 2002;Murdock et al., 2012). The higher level of oxygen in unshelled groundnuts may be explained by the bulkiness of the shells that create more air space compared to shelled groundnuts, which are more compact and contain less free airspace.
Germination of shelled and unshelled groundnuts kept in PICS bags for 6.7 months did not change e there was only a slight reduction of less than 3%. However, storage of groundnuts in woven bags caused germination reductions of up to 64.8% for shelled groundnuts. Nautiyal (2002) reported a reduction of 73% in germination when shelled groundnuts were infested by T. castaneum. These findings are consistent with the results of Sudini et al. (2015) who reported that PICS bags protected groundnut seed viability and seed weight under artificial infestation with C. serratus. Similar results have been noted on naturallyinfested stores of Bambara groundnut and maize (Baoua et al., 2014a(Baoua et al., , 2014b. The loss of germination recorded in the control treatments was almost certainly due to seed-embryos destruction by the insects. PICS bags performed better than Supergrain bags (another type of hermetic bag available to farmers in developing countries) for storing groundnuts (Harish et al., 2014). Supergrain bags enabled the development of C. serratus with a weight loss of 66.9% and 75.9% after 6 months of storage for shelled and unshelled groundnuts, respectively. We observed no perforations or other damage to the PICS HDPE liners. This suggests that the bags can be reused for two or more seasons, decreasing their overall cost and increasing the benefit to farmers.
In summary, naturally infested shelled and unshelled groundnuts can be safely stored in PICS bags, which arrest insect population growth and prevent associated grain degradation without affecting germination. Storing shelled groundnuts in PICS appear to be cost-effective.


s2fieldsofstudy: Biology Medicine

text:
Podocyte Autophagy: A Potential Therapeutic Target to Prevent the Progression of Diabetic Nephropathy

Diabetic nephropathy (DN), a leading cause of end-stage renal disease (ESRD), becomes a worldwide problem. Ultrastructural changes of the glomerular filtration barrier, especially the pathological changes of podocytes, lead to proteinuria in patients with diabetes. Podocytes are major components of glomerular filtration barrier, lining outside of the glomerular basement membrane (GBM) to maintain the permeability of the GBM. Autophagy is a high conserved cellular process in lysosomes including impaired protein, cell organelles, and other contents in the cytoplasm. Recent studies suggest that activation of autophagy in podocytes may be a potential therapy to prevent the progression of DN. Here, we review the mechanisms of autophagy in podocytes and discuss the current studies about alleviating proteinuria via activating podocyte autophagy.

Introduction
Diabetes mellitus (DM) has been one of the global health issues. According to the report from the International Diabetes Federation, the number of patients with DM will increase to 205 million in 2035 than in 2014. Diabetic nephropathy (DN), a serious chronic complication of DM, is a leading cause of end-stage renal disease (ESRD). One significant clinical feature of DN is the appearance of urinary protein, defined as "albuminuria." Structural changes of the glomerular filtration barrier are detected in the diabetic patients with albuminuria, including glomerular endothelial cell injury, the loss of podocytes, glomerular basement membrane (GBM) thickening, and mesangial expansion [1,2]. Apart from GBM dysfunction, the accumulation of advanced glycation end products (AGEs), oxidative stress, and the activation of the renin-angiotensin system (RAS) also contribute to the decline in renal function [1,[3][4][5][6][7].
Based on the pathologic alterations in the kidney, DN is classified into four groups: class I includes GBM thickening, class II consists of mild (IIA) to severe (IIB) mesangial expansion, class III includes nodular glomerulosclerosis, and class IV represents developed DN which is characterized with over 50% global glomerulosclerosis and podocyte deficiency [8,9]. Among these four categories, the kidney may also exhibit arteriolar hyalinosis, glomerular capillary subendothelial hyaline, and arteriosclerosis. Present therapies are mainly focusing on the way to reduce the levels of blood glucose and blood pressure to normal, and most of them alleviate albuminuria via suppressing the RAS activity [10]. Nevertheless, considering the elevation of diabetic kidney diseases, further studies in a pathogenetic mechanism for DN are needed to find new approaches to treat DN. Recently, a number of reports have demonstrated that autophagy is involved in the pathogenesis of diabetesrelated podocyte injury. In this review, we will make a summary on the role of autophagy in this process and the mechanisms involved.

Autophagy
Autophagy (from the ancient Greek meaning "self-eating") is a highly conserved cellular process that delivers protein and other impaired cell organelles to lysosomes for degradation and recycle to maintain intracellular homeostasis. Christian de Duve first referred autophagy in 1963 [11]. Subsequent studies focused on the regulatory mechanisms of autophagy and its effects on human health and disease.
On the basis of different ways of transporting intracellular constituents to lysosomes, autophagy is divided into three types: macroautophagy, microautophagy, and chaperonemediated autophagy [12]. Macroautophagy and chaperonemediated autophagy are through autophagosomes and chaperone complex, respectively, while constituents are delivered to lysosomes directly in microautophagy [13,14]. In this review, macroautophagy (hereafter referred to as autophagy) is mainly investigated among these three types. In terms of different types of degraded substrates, autophagy was also divided into selective and nonselective autophagy. Degradation of some impaired organelles, lipophagy, or xenophagy is involved in selective autophagy, whereas deficient nutrient-induced autophagy is considered the nonselective type [15][16][17].

Podocyte Autophagy in Diabetic Nephropathy
Studies have demonstrated that autophagy is renoprotective in acute kidney injury, obstructive nephropathy, diabetic nephropathy, and other renal diseases [22]. Podocytes are highly differentiated epithelial cells lining the outer aspect of the GBM with interdigitating foot processes, and the slit diaphragms between foot processes play a role in substance filtration. Podocyte injury including foot process fusion and slit diaphragm alteration results in abnormal permeability of the GBM, terminally leading to albuminuria. Autophagy controls the quality of the cytoplasm, via degrading proteins, peroxidases, and damaged organelles that complicate the recycle of organelles, and then maintains the homeostasis of intracellular environment [23,24]. The self-repaired feature of autophagy is important in the anaphase cells such as neurocytes and podocytes, which have a restricted capacity in differentiation and proliferation [25]. The previous studies explored the mechanisms of podocyte autophagy in DN and suggested that activated podocyte autophagy has an effect on DN through an Atg12-Atg5 conjugation system, mTOR, adenosine 5′-monophosphate-(AMP-) activated protein kinase (AMPK), and oxidative stress as well as vascular endothelial growth factor.
3.1. Atg12-Atg5 Conjugation System in Podocyte Autophagy and Diabetic Nephropathy. Atg12 is a ubiquitin-like protein involving in autophagosome formation. Autophagy activation needs the conjugation of Atg12 to Atg5, which is stimulated by Atg7 and Atg10, and then promotes Atg8 and lipid phosphatidylethanolamine conjugation in the cytoplasm [26]. The activation of the Atg12-Atg5 conjugation system promotes the production of autophagosome and then activates podocyte autophagy. Currently, Liu et al. demonstrated that β-arrestins, a negative adaptor of G protein-coupled receptors (GPCRs), aggravate podocyte injury through autophagy inhibition in DN [27]. They found that β-arrestins suppressed podocyte autophagy via downregulating Atg12-Atg5 conjugation, which is induced by enhancing the interaction between β-arrestins and Atg7. Therefore, modulation of this pathway may be a novel therapeutic approach for treating patients with DN.
3.2. mTOR Signaling Pathway in Podocyte Autophagy and Diabetic Nephropathy. Mammalian target of rapamycin (mTOR) is essential to cell growth regulation, and activation of mTOR suppresses autophagy. Deficient nutrients (such as growth factor or amino acid deficiency) in the cytoplasm activate autophagy by suppressing the expression of mTOR. After inhibition, mTOR not only can activate the formation of class III phosphatidylinositol 3-kinase (PI3K) complex and the unc-51-like kinase (Ulk) 1 complex but also inhibit the activity of ribosome protein subunit 6 kinase 1 (S6K1) [28][29][30][31]. In the upstream of mTOR, there are two separated protein kinases, phosphatidylinositol 3-kinase I (PI3K-I)/ protein kinase B and AMP-activated protein kinase, which are regulated by different conditions [32].
In contrast, the class I PI3K regulatory subunit p58 is bonded to the catalytic subunit p110 and then activates the Akt/ mTOR signaling pathway [39,40] by promoting phosphatidylinositol 3,4,5-triphosphate. Therefore, it seems that class I PI3K inhibits autophagy while class III PI3K activates it. The activation of class I PI3K is triggered by insulin or growth factors to interact with insulin receptors or tyrosine kinase receptors, which are the members of transmembrane receptors existing on the membrane of podocytes and then activates Akt/PKB. Then, the downstream tuberous sclerosis complex 1 and 2 (TSC1/2) proteins will be inhibited by PKD1 and the production of Akt/PKB activation. In the end, podocyte autophagy is suppressed by the activation of mTOR. Recent studies have emphasized the relationship between DN and nutrient-dependent pathways, involving the mTOR signaling pathway. In the models of diabetic nephropathy, especially the type 1, insulin resistance blocks the phosphorylation of Akt/PKB and then activates mTOR by increasing the expression of Rheb (Ras homolog enriched in brain). Thus, insulin resistance suppresses podocyte autophagy through increasing the activity of mTOR.

AMP-Activated Protein Kinase (AMPK).
As an essential regulator in energy metabolism, AMPK is an enzyme consisted of three proteins (α, β, and γ) [41]. AMPK can be activated by an increase in Ca 2+ concentration in the cytoplasm [42,43] and the stimulation of numerous hormones, adipokines, and cytokines. In addition to these, the ratio of an intracellular AMP/ATP decrease also activates AMPK. Nutrient starvation induced the activation of AMPK. In the condition of ATP deficiency, the downstream TSC1/2 is activated by AMPK, then inhibits Rheb, and finally enhances autophagy by suppressing mTOR activation ( Figure 1). Recently, Jin et al. suggested that berberine alleviated high glucose-induced apoptosis of podocytes in mouse via increasing the activity of AMPK [44]. They showed that the expression of p-AMPK in a high-glucose (HG) group was lower and the expression of p-mTOR was higher in the HG group compared with the control group, while these results were conversed by berberine administration.
Mechanical stress induced by the renin-angiotensin system is considered a major damage factor in podocytes of DN. Spironolactone, a common diuretic, is generally used to treat heart failure, edema, or Conn's syndrome. The study from Li et al. demonstrated that spironolactone has renoprotective effects on activating autophagy through blockage of the mTOR signalling pathway in podocytes under mechanical stress [45]. They used the Flexercell FX-5000TM Compression System to establish the animal model of DN and found that the expressions of p85-PI3K, p-AKT, and p-mTOR were significantly increased compared with those of the control group. After administration of spironolactone for 48 h, the levels of p85-PI3K, p-AKT, and p-mTOR were markedly decreased, which are in accordance with the results in the group by PI3K inhibition. Thus, spironolactone might be a new therapy of DN.
Rapamycin is a new immunosuppressive drug of macrocyclic lactone, which was first found in a soil bacterium in 1965 [46]. After that, researchers suggested that rapamycin has antifungal effects as well as anti-T cell activity in succession [47]. Furthermore, rapamycin is a selective inhibitor of mTOR [48]. Rapamycin binding to immunophilins, such as FKBP12 (FK binding protein, 12 kDa), forms an FKBP12-rapamycin complex. The FKBP12-rapamycin complex suppresses the expression of mTOR through phosphorylation of mTOR and then activates autophagy. However, the number of clinical trials of rapamycin in DN is less; further studies are needed to clarify the renoprotective property of rapamycin in DN.

Reactive Oxygen Species (ROS) in Podocyte Autophagy
and Diabetic Nephropathy. Besides insulin and nutrition starvation, intracellular metabolism alternations are also related to the pathogenesis of DN, involving the increase in reactive oxygen species (ROS). Several studies have shown that ROS are the most common factors in activating podocyte autophagy. An increase in ROS production activates PKR-like kinase (PERK), which then oxidizes Atg4 proteases via eIF2a phosphorylation, subsequently promotes the level of proteolytic mature LC3, and prevents mTOR activation [49]  that podocyte autophagy was activated by upregulating the generation of mitochondrial ROS after exposing to high glucose for 24 hours [50]. Meanwhile, podocytes exposed to angiotensin II (ANGII) also increased the generation of ROS and promoted autophagy activation [51]. However, the membrane of the mitochondrion is damaged by excessive ROS generation in the mitochondrion, and ROS releasing into the cytoplasm may cause damage to other organelles. Since the function of autophagy targeting and degrading injury organelles is selective, the augmentation of ROS will be limited [52]. Chronic exposure to high-glucose condition leads to autophagy insufficiency and subsequently causes lysosomal dysfunction and podocyte apoptosis, finally resulting in diabetic nephropathy [53]. Therefore, reduction of ROS generation is a potential therapeutic approach for preventing the development of DN.

Vascular Endothelial Growth Factor (VEGF) in Podocyte
Autophagy and Diabetic Nephropathy. In the early phases of animals or patients with DN, the level of VEGF has been shown to be increased in the kidney. Several studies have suggested that elevation of VEGF is associated with the increase in the glomerular permeability, then resulting in proteinuria [54].VEGF is considered to be a promoter of angiogenesis and synthesized mainly by the podocytes. VEGF-A, as one member of a VEGF family, has a negative effect on glomerular endothelial cell (GEC) glycocalyx through the early stages of DN, and this effect can be reversed by VEGF-A 165b , an inhibitory isoform of VEGF-A, finally ameliorating proteinuria [55]. Autophagy has been reported to prevent angiogenesis [56,57]. Miaomiao et al. found that high glucose enhanced the level of VEGF, whereas this elevation is downregulated by autophagy activation via rapamycin, an inhibitor of mTOR [58]. Yang [59] and Liu et al. [60] also demonstrated that the increase in autophagosome inhibits angiogenesis.

Conclusion
According to the International Diabetes Federation, the global diabetes prevalence will increase from 8.3% in 2014 to 10.1% in 2053. As a serious global health issue, it is urgent to find potent therapies to treat diabetes and its complications, especially diabetic nephropathy. The previous studies have shown the activation of autophagy in podocytes via inhibiting the expression of mTOR and alleviating albuminuria in DN. Meanwhile, autophagy activation also decreased the expression of VEGF and subsequently prevented the progression of DN. Although studies have suggested that podocyte autophagy is a renoprotective process in lysosome, DN is an extremely complex complication. Further investigations are needed to elucidate the role of autophagy in podocyte injury induced by DN and discover the autophagy-based therapies for the treatment of DN.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
Na Liu and Liuqing Xu are co-first authors.