s2fieldsofstudy: Biology Medicine

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Immune cell distribution and immunoglobulin levels change following sciatic nerve injury in a rat model.

Objective(s): To investigate the systemic and local immune status of two surgical rat models of sciatic nerve injury, a crushed sciatic nerve, and a sciatic nerve transection Materials and Methods: Twenty-four adult male Sprague-Dawley rats were randomly divided into three groups: sham-operation (control group), sciatic nerve crush, and sciatic nerve transaction. Sciatic nerve surgery was performed. The percentage of CD4+ cells and the CD4+/CD8+ratio were determined by flow cytometry. Serum IgM and IgG levels were analyzed by ELISA. T-cells (CD3) and macrophages (CD68) in sciatic nerve tissue sections were identified through immunohistochemistry. Results: Compared to sham-operated controls, in rats that underwent nerve injury, the percentage of CD4+ cells and the CD4+/CD8+ ratio in the peripheral blood were significantly decreased 7 days after surgery, serum IgM levels were increased 14 days after surgery, and serum IgG levels were increased 21 days after surgery. There were a large number of CD3+ cells and a small number of CD68+ cells in sciatic nerve tissue sections 21 days after surgery, indicating T-cell and macrophage activation and infiltration. Local IgG deposition was also detected at the nerve injury site 21 days after surgery. Conclusion: Rat humoral and cellular immune status changed following sciatic nerve injury, particularly with regard to the cellular immune response at the nerve injury site.

Introduction
Peripheral nerve injury is an under-appreciated clinical problem, even though it is more common than injury to the central nervous system. Indeed, repair and regeneration of peripherally injured nerves is an extremely complex process (1), which includes adhesion, the extracellular matrix, regulation of neurotrophic factors, formation and extension of the growth cone, synthesis and release of neurotransmitters, and neuron remodeling. In addition, inflammation, inflammatory cells and their products, and major histocompatibility complex class II (MHC II) antigens have a very important role in the degeneration and regeneration of axons (2,3). As such, the success of peripheral neuroregeneration after trauma or disease is limited, even when compared to neuroregeneration after damage to the central nervous system (4).
Evidence suggests that neuro-immune interacttions take place in response to disease and trauma in the nervous system (5). Following peripheral nerve injury, a series of immunological responses occurs (3). Both neuroprotective and neurodestructive effects of the immune system have been described, although how these contradictory effects are regulated has yet to be determined (6). Importantly, the immune response after peripheral nerve injury may cause secondary damage to neurons and inhibit their repair and regeneration. Some studies show that peripheral nerve damage stimulates the innate immune response and the production of a variety of cytokines and inflammatory mediators, which induce non-specific nerve damage (7)(8)(9). Elucidating the crucial immune elements that regulate peripheral nerve injury may inform the development of novel therapeutic strategies that suppress immune reactions, promote nerve repair and regeneration, and restore normal function following nerve injury.
To further understand the role of the humoral and cellular immune response in regulating peripheral nerve damage, we used rat models of sciatic nerve injury. We focused on characterizing CD4 + cells, the CD4 + /CD8 + ratio, and local immuneglobulin (Ig) G and IgM levels in the peripheral blood and cellular infiltrates at the nerve injury site. The percentage of CD4 + cells and the CD4 + /CD8 + ratio in the peripheral blood are good indicators of overall immune status (10). The local expression of CD3 and CD68, and the deposition of immunoglobulin following sciatic nerve injury provide evidence of interactions between the immune response during nerve regeneration.

Animals
This study was approved by the Committee on the Ethics of Animal Experiments of the Peking University People's Hospital, China. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Twenty-four adult male Sprague-Dawley rats (mean body weight, 275 g; range, 250-300 g) from the Animal Center of the Military Academy of Medical Sciences were used in this study. The rats were maintained under standard pathogen-free laboratory conditions on a 12 hr light/dark cycle with free access to pellet food and water. Rat care and all experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Chinese National Committee for the Use of Experimental Animals for Medical Purposes, Beijing Branch.

Sciatic nerve surgery
The rats were randomly divided into three groups: sham-operation (control group) (n= 8), sciatic nerve crush (n= 8), and sciatic nerve transection (n= 8). The rats were anesthetized with sodium pentobarbital (30 mg/kg) by intraperitoneal injection. All of the surgical procedures were performed under aseptic conditions. The sciatic nerves of the rats in the sham-operated control group were exposed, but no deliberate injury was performed. In the sciatic nerve crush group, the left sciatic nerve of each rat was clamped for 1 min using pincers with a 2 mm width (11). The sciatic nerve transection in the third treatment group was performed by severing the left sciatic nerve with a scalpel (12). The stump of the nerve was sutured with 3 to 4 stitches of 9-0 silk thread by neurorrhaphy.

Percentage of CD4 + cells and CD4 + /CD8 + ratio in peripheral blood
Blood was collected through the orbital venous plexus of each rat at 7, 14, and 21 days post-surgery. Peripheral blood samples were obtained from individual rats, and the percentage of CD4 + cells and the CD4 + /CD8 + ratio were determined by flow cytometry using a FACS Calibur instrument, according to the manufacturer's instructions (BD Biosciences).

Enzyme-linked immunosorbent assay (ELISA) analysis of serum antibodies
The concentrations of serum antigen-specific IgG and IgM in individual animals were analyzed by ELISA, according to the manufacturer's instructions (Bethyl, USA). Individual serum samples at 1:100-1:500 dilutions were tested in triplicate and incubated at 37 °C for 1 hr. Subsequently, the bound antibodies were incubated with horseradish peroxidase (HRP)conjugated goat anti-rat IgG (1:100) or HRP-conjugated goat anti-rat IgM (1:100) (Bethyl, USA) at 37 °C for 1 hr. After washing, the bound HRP-conjugated secondary antibodies were detected with tetramethylbenzidine substrate. The reaction was stopped by adding 50 μl/well of 1 M H2SO4, and the optical density was measured at 450 nm.

Immunohistochemistry
The rats were euthanized 21 days after surgery. The left sciatic nerve was dissected, fixed in 4% paraformaldehyde for 48 hr, conventionally dehydrated, cleared, embedded in paraffin, and sectioned into 4 μm sections. The sections were dewaxed and incubated in 3% H2O2 at room temperature for 10 min to inactivate endogenous peroxidase. After washing 3 times, the sections were microwaved for 10 min in 0.01 M citrate buffer (pH= 6.0) and washed 3 times.
Primary antibodies were diluted with phosphatebuffered saline (PBS) and 5% goat serum. CD3 and CD68 (1:500, Abcam, USA) and IgG (1:200, PLlabs, USA) antibodies were added in sequential treatments, according to the manufacturer's instructions. After overnight incubation and PBS washing, a secondary antibody conjugated with biotin was added. The sections were incubated with HRPconjugated streptavidin at room temperature for 30 min. The antigens were visualized with 3,3'diaminobenzidine. The sections were washed with double-distilled water, air-dried, and fixed with neutral balsam.
Sections were observed under a Leica DM6000B microscope (Leica, Germany), and the images were processed with IPP6.0 software. T-cells (CD3) and macrophages (CD68) were quantified on 4 sec/rat (n= 8 rats/group) at the region with cell infiltration or at the sciatic nerve injury site.

Statistical analysis
The experimental data are expressed as mean±standard error of the mean (SEM). Statistically significant differences between the groups were determined by one-way analysis of variance

Percentage of CD4 + cells and CD4 + / CD8 + ratio in the peripheral blood
At 7 days post-surgery, there were 60.84±2.49% CD4 + cells in the peripheral blood of rats that underwent sciatic nerve crush injury and 58.5±3.92% CD4 + cells in rats that received sciatic nerve transection. These were significantly lower than the percentage of CD4 + cells in the peripheral blood of the sham-operated control group (65.94±2.66%, P<0.05). The ratio of CD4 + /CD8 + in the peripheral blood was also significantly lower in the rats that underwent a sciatic nerve crush injury (2.04±0.22) or sciatic nerve transection (1.97±0.39) compared to the shamoperated control group (2.5±0.27) (P< 0.05). At 14-and 21 days post-surgery, there were no statistically significant differences between the sciatic nerve injury groups and sham-operated controls in the percentage of CD4 + cells or the CD4 + /CD8+ ratio in the peripheral blood (P>0.05) (Figure 1).

Serum levels of IgG and IgM
At 7 days post-surgery, there were no statistically significant differences in serum IgG or IgM levels between the sciatic nerve crush, sciatic nerve transection, and sham-operated control groups. At 14 days post-surgery, serum IgM levels were significantly increased in the rats that underwent sciatic nerve injury compared to the sham-operated control group (P<0.05); there were no significant Error bars represent the SEM of three independent experiments. Asterisks (*) indicate statistically significant differences compared with Group 1. Serum IgM was significantly elevated at 14 days, but not 7 days or 21 days postsurgery in Groups 2 and 3 compared to group 1. Serum IgG was significantly elevated 21 days post-surgery in Groups 2 and 3 compared to Group 1 differences in serum IgG levels. At 21 days postsurgery, serum IgM levels had decreased in the rats with sciatic nerve injury, and there were no statistically significant differences between the groups. In contrast, serum IgG levels were significantly increased in the rats with nerve injury; both the sciatic nerve crush and the sciatic nerve transection groups had significantly higher serum IgG levels compared to the sham-operated control group (P<0.05; Figure 2).

Hematoxylin and eosin (H&E) staining
At 21 days post-surgery, sciatic nerve tissue sections were collected from each group and stained with H&E. In the sham-operated control group, medullated fibers appeared to be arranged normally, and there was no inflammatory cell infiltration ( Figure 3, group 1). In the rats with sciatic nerve injury, the numbers of medullated fibers were reduced, axons were inflamed and disarranged, and there were a large number of infiltrated lymphocytic cells. In addition, a portion of the nerve fibers had disintegrated and was engulfed by phagocytes ( Figure 3, group 2, group 3).

Immunohistochemical staining
At 21 days post-surgery, immunohistochemical staining of sciatic nerve tissue sections revealed a large number of CD3+ cells and a small number of CD68+ cells in rats with sciatic nerve injury (Figure 4). In contrast, CD3+ or CD68+ cells were not detected in the sham-operated control group. There were significant differences in the number of CD3+ and CD68+ cells in infiltrates between the rats with sciatic nerve injury and the sham-operated control group (P<0.01; Table 1). Although the numbers of CD3+ and CD68+ cells in the rats that underwent sciatic nerve transection were higher compared with rats with sciatic nerve crush injury, this difference was not statistically significant (P>0.05). These results indicate that a large number of lymphocytic cells had been recruited locally at the sciatic nerve injury site.

Qualitative analysis of immunoglobulin deposition
At 21 days post-surgery, IgG deposition ( Figure 5, group 1) was not detected in sciatic nerve tissue sections in the sham-operated control group. However, there was sporadic IgG deposition along the nerve fiber bundle in rats from the sciatic nerve crush group and a moderate amount of IgG deposition along the nerve fiber bundle in rats with sciatic nerve transection ( Figure 5, group 2, group 3).

Discussion
In this study, we used two rat models of sciatic nerve injury to investigate the humoral and cellular immune response following peripheral nerve damage. In both the sciatic nerve crush and sciatic nerve transection rat models we observed systemic and local changes in immune status after nerve injury and found that humoral and cellular immune responses were altered. Seven days after nerve injury, the percentage of CD4 + cells and the CD4 + /CD8 + ratio in the peripheral blood were significantly lower in rats with nerve damage compared with the sham-operated control rats. These findings indicate that rats have a low immune status after nerve injury, which may result in an underactive and poor performing immune system. This is consistent with the period of immunosuppression seen in patients after trauma (13). The period of low immune status in the rats was transient and T cell levels returned to normal 14 days after nerve injury.
It is well known that IgG and IgM are the major immunoglobulins in blood serum (14,15). IgG is a product of the initial response to infection and secondary immune response and memory and therefore has important immune effects. IgM can activate complement more effectively than IgG, and serum levels of IgM begin to elevate when the humoral immune response is initiated. In our rat models of sciatic nerve injury, we observed significantly increased levels of serum IgM 14 days after nerve injury, which normalized by day 21. Levels of serum IgG increased only after 21 days; the delay may be because the generation of IgG in the blood requires antigen uptake, processing, and presenting. The systemic immune response seen in our rat models was likely the result of a breakdown in the blood-nerve barrier following nerve injury and the subsequent release of nerve antigens into the blood circulation, which stimulates immune cells and generates specific antibodies (16). We also observed IgG deposition locally at the sciatic nerve injury site, IgG deposition was not observed in the shamoperated controls. This further substantiates the suggestion that specific antibodies act on nerve cells after their release into the circulation to propagate an immune response.
In this study, we hypothesized that differences in the degree of nerve damage might be reflected by the cellular immune response at the nerve injury site. However, we found no significant difference in the percentage of CD4 + cells, the CD4 + /CD8 + ratio, and serum IgG and IgM levels in rats that had undergone sciatic nerve crush injury and sciatic nerve transection. This may be due to the acute nature of the injuries. A study of chronic lumbar disc herniation patients revealed significant differences in peripheral blood counts of CD4 + cells and the CD4 + /CD8 + ratio in patients stratified according to the degree of lower back pain (17). In contrast, CD3+ and CD68+ infiltration was less in the rats that had undergone sciatic nerve crush injury compared to the rats with a sustained sciatic nerve transection. CD3 is required for T cell activation and CD63 is expressed on the surface of monocytes/macrophages. Macrophages are able to take up, process, and present antigens to T lymphocytes. Macrophages also express MHC II antigens, which have a crucial role in the regulation of the immune response after peripheral nerve injury (18). The enhanced expression of MHC II antigen can cause the synthesis and deposition of immunoglobulin and immune cell infiltration, which has an inhibitory effect on nerve regeneration.
The present study showed that Wallerian degeneration of nerve fibers occurred after peripheral nerve injury. Wallerian degeneration refers to the sequence of events that occurs following nerve transection. Wallerian-like degeneration refers to similar events that occur in damaged axons after blunt or crush injury. Secondary damage to the neurons caused by the immune response after peripheral nerve injury can aggravate Wallerian degeneration and inhibit the repair and regeneration of peripheral nerves (19,20).

Conclusion
In the present study, we found that during the 21 days following sciatic nerve injury in rats, there were changes in both the humoral and cellular immune systems, especially the cellular immune response that locally affects the injured nerve. Alterations in the humoral and cellular immune systems varied during the 21 days post-injury, likely reflecting different stages of activation. The significance of such changes in terms of the rate and efficacy of peripheral nerve recovery remains unclear. Furthermore, how signals of these changes in immune system status are relayed to the nervous system and translated requires further investigation. Additional study is needed to understand the mechanism of these processes to provide an adequate theoretical basis for the clinical application of immune system modulating drugs following peripheral nerve injury.


extfieldsofstudy: Chemistry Medicine

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Importance of the residue 190 on bactericidal activity of the bactericidal/permeability-increasing protein 5

The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5.

IntroductIon
Gram-negative bacterial infections can result in severe pathologic changes including fever, disseminated intravascular coagulation and multisystem organ failure [1,2]. The major virulence factor of the Gram-negative bacteria has been identified as lipopolysaccharides (LPS), a glycolipid structural component on the bacterial cell wall [3][4][5]. Release of LPS into the circulation induces a systemic inflammatory response [6]. Therefore, a promising therapeutic agent for Gram-negative bacteria infections has both antibacterial and antiendotoxic properties.
The bactericidal/permeability-increasing protein (BPI), a multifunctional cationic protein with 55 to 60 kDa, is originally found in granules of polymorphonuclear leukocytes, and more recently, is detected on the surface of neutrophils and in eosinophils [7][8][9]. Extensive studies show that BPI exhibits powerful antibacterial activity, largely due to the high affinity of BPI for the conserved lipid moiety in LPS [7,10]. Binding of BPI to Gramnegative bacteria would initiate growth arrest, increase outer membrane permeability, selectively activate endogenous bacterial phospholipases and ultimately cause bacterial death [11,12]. BPI also forms complex with cell-free LPS and consequently inhibits all LPS-induced host immune responses. Therefore, the antibacterial and antiendotoxin activities of BPI attract high interesting for the treatment of Gram-negative bacteria infections [13,14].
Structural characterization of BPI has provided insight into its function. The crystal structures of human BPI show a boomerang-shaped molecule formed by two similar domains, N-terminal domain and C-terminal domain [15,16]. A high proportion of basic residues are located at the surface and forms positively charged tips on N-terminal domain, resulting in high affinity of BPI with negatively charged LPS. This character is well consistent

Research Paper
with observations that N-terminal region carries all the antibacterial and almost endotoxin-neutralizing activities of BPI [17][18][19]. For C-terminal domain, it is essentially neutral and only shows limited LPS-binding ability [20]. Few effects, however, have been directed toward targeting the precise residues or regions of BPI, where they contribute critically to the bactericidal and endotoxin neutralization actions.
Recently, we identified a putative causative mutation, replacing Asp190 with alanine in the mBPI5 gene (GenBank Accession No. NM_144890) that might be responsible for the male infertility of Blind sterile (bs) mice (data not shown). In this report, we extend our studies to investigate the effect of Asp190Ala mutation in mBPI5 on its antibacterial activity. Wild-type and this mutate of mBPI5 were expressed in E. coli as inclusion bodies, refolded by high concentration of urea and purified using His-tag. Antibacterial assay and liposome binding assay were performed to measure their corresponding changes caused by introduction of alanine on position 190. A structural model of mBPI5 was constructed to explore the mechanism underlying these changes.

The expression and purification of mBPI5 and Asp190Ala mutant
Of great interests as novel antibiotics, large quantities of highly purified BPI are required to meet the needs of basic research and clinical trials. Among the systems available for heterologous protein production, E. coli has been the most widely used host. The coding sequences of mBPI5 and Asp190Ala mutant were cloned into the expression plasmid pET32a. mBPI5 and its mutant produced in E. coli strain SHuffle were expressed as inclusion bodies such that its lethal effect towards the host could be masked and protect them from proteolytic degradation. Inclusion bodies of mBPI5 were solubilized by the use of a high concentration of urea (8M) along with β-mercaptoethanol, yielding an appreciably high recovery of 26%. High quality purification of mBPI5 and Asp190Ala mutant could be achieved by one-step nickelaffinity chromatography ( Figure 1).

The effect of Asp190Ala mutation on antibacterial activity of BPI
BPI has potent bactericidal activity against a wide variety of Gram-negative organisms. Two representative Gram-negative clinical isolates, E.coli J5 and P.aeruginosa that have high intrinsic resistance to antibiotics, were chose to assess the effect of Asp190Ala mutation on the antibacterial activity [25].
As shown in Figure 2, both mBPI5 and Asp190Ala mutant suppressed the growth of E.coli J5 and P.aeruginosa in a concentration-dependent manner. Under relatively low concentrations (< 10 nM), the antibacterial activity of Asp190Ala mutant against two Gram-negative isolates was similar to that of wild-type. However, compared with wild-type mBPI, Asp190Ala mutant had a stronger effect on suppressing Gram-negative bacteria growth at higher concentrations. These results suggested that the introduction of alanine at residue 190 significantly enhanced the antibacterial activity of mBPI5. (Figure 2) The effect of Asp190Ala mutation on the binding affinity of mBPI5 to liposome BPI's cytotoxic activity that is selectively manifest toward Gram-negative bacteria has been attributed to its high affinity for the lipid moiety of LPS or endotoxin [7,10]. The negatively charged liposome has been used frequently for mimicking the negatively charged bacterial membrane [26]. The effect of Asp190Ala mutation on binding affinity was measured by using affinity precipitation assay.
As shown in Figure 3, Asp190Ala mutant almost existed in the form of bound with lipid, whereas native mBPI5 bound weakly to liposomes. The ratio of the amount of protein binding to liposome to the amount of protein in supernatant was 1.03 and 8.46 for mBPI5 and Asp190Ala mutant, respectively, that is, this ratio of Asp190Ala mutant increased about 8 folds compared with that of mBPI5. These data suggested that the enhanced binding affinity to lipid accounted for the increased antibacterial activity of Asp190Ala mutant. (Figure 3).

The structural analysis of mBPI5
In order to shed light on mechanism by which introduction of alanine at position 190 greatly enhanced its antibacterial activity and affinity for lipid, a structural model of mBPI5 was required. Since no suitable structure templates that shared high sequence similarity with mBPI5 could be found, threading method (I-TASSER) was used to predict its structure, by which a library of representative structures was searched for structure analogs to mBPI5, mBPI5 was aligned with structure templates by secondarystructure enhanced profile-profile threading alignment, and structure models of mBPI5 were generated by iterative structural assembly simulations. The best model was selected based on its C-score, TM score and RMSD value.
Just like that of human BPI, the dimensional structure of mBPI5 displayed boomerang-shaped molecule formed by two domains ( Figure 4A). The N-terminal and C-terminal domain shared a similar two-layer α/β structure, a unique fold only found in BPI family. Asp190 was located on the surface of the N-terminal domain. Closer examination showed that the negatively charged carboxylate of Asp190 formed salt linkages with the positively charged guanidinium at the end of the side chain of arginine 188 ( Figure 4B). When replacing Asp190 with alanine, these electrostatic interactions were broken so that the positively charged side chain of Arg188 was free to move and established tighter contacts with negatively charged LPS.
Besides Arg188, other five basic residues, including Lys96, Lys102, Lys103, Lys187 and Lys199, were folded into the proximity of Asp190, that is, a high proportion of basic residues was concentrated in the vicinity of Asp190 ( Figure 4B). Therefore, the presence of negatively charged Asp190 could lower and disturb electrostatic interactions between these basic residues and LPS.

dIscussIon
Although most Gram-negative bacteria are susceptible to antibiotics, therapies are needed to counteract the inflammatory effects of killed bacteria and/ or their endotoxins [6]. Among the potential therapeutic agents for Gram-negative sepsis, BPI is especially promising since it has both antibacterial and antiendotoxic properties. In this study, we define the critical role of residue 190 in antibacterial and antiendotoxic activities. We found that the replacement of Asp190 with alanine  Both mBPI5 and Asp190Ala mutant can suppress E. coli J5 and P.aeruginosa in a concentration-dependent manner. Under relatively low concentrations (< 10 nM), the antibacterial activity of Asp190Ala mutant against two Gram-negative isolates is similar to that of wild-type. However, Asp190Ala mutant has a stronger effect on suppressing Gram-negative bacteria growth at higher concentrations. All data are shown as mean ± SD (*p < 0.05; **p < 0.001); bar SD. www.impactjournals.com/oncotarget  can greatly enhance the antibacterial activity of mBPI5 to E.coli J5 and P. aeruginosa. The enhanced antibacterial activity of Asp190Ala mutant is brought about by a marked increase in affinity to lipids. Constructed structure of mBPI5 suggested that replacing Asp190 with alanine could avoid the unfavorable contacts of negatively charged Asp190 with negatively charged LPS. These results could improve our understanding of the structure-function relationship of BPI, which paves way for site-directed mutagenesis experiments to modify BPI for clinical applications.

Protein expression, refolding and purification
The recombinant mBPI5 and its mutant were expressed in E. coli strain SHuffle. Cells were grown at 37°C in LB medium containing 50 μg/ml Ampicillin to an OD600 of 0.6, and were then induced by the addition of 0.1 mM isopropyl β-D-thiogalactoside (IPTG) at 16°C for 12 h. Cells were harvested and re-suspended in buffer (25 mMTris-HCl pH 8.0, 300 mM NaCl), disrupted by sonication and centrifuged at 12000 g for 20 min at 4°C. Recombinant protein was exclusively detected in the precipitates. BPIs inclusion bodies were resuspended in 20 ml solubilization buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 8 M urea, pH 8.0), incubated by stirring for 2 h at room temperature, and then centrifuged at 12000 g for 20 min. The supernatant was applied to a nickel affinity column(Qiagen WorkBeads TM 40 Ni #40650003) equilibrated with the same buffer. Removal of contaminating proteins and initial refolding were carried out on the column by exchanging to nondenaturing buffer conditions with a linear 8-0 M urea gradient before elution of the bound protein.The recombinant BPIs was eluted with elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 300 mM imidazole, pH 8.0) and concentrated by ultrafiltration (Amicon #UFC500308),The eluted products were determined by Bradford protein assays (Bio-Rad Protein Assays Kits #5000001) analyzed by 15% reduced and non-reduced SDS-PAGE.

Bactericidal activity assay
Cells were grown overnight in LB medium and then in galactose-free triethanolamine-buffered medium at 5 × 10 8 to 10 × 10 8 cells per mL corresponding to midto late-logarithmic phase. The culture was harvested and adjusted to an OD600 of 0.5 in physiological saline. The bacterial suspension (5 × 10 6 cells) was incubated for 30 min at 37°C with various concentrations of mBPI5, Asp190Ala mutant or buffer control in a medium consisting of 10% Hanks balanced salt solution (GIBCO #14170112), 40 mM Tris-HCl pH 7.5, and 0.1% Casamino Acids in a total volume of 200 μL. MgCl 2 was added, when desired, at a final concentration of 100 mM to the mixture described above. The effect of mBPI5 on bacterial viability was assessed by plating dilutions of the cells on nutrient agar plates and enumerating bacterial colonies after overnight incubation at 37°C.

Liposome co-sedimentation assay
Liposome co-sedimentation assay was performed as described previously [21]. Lipid mixes (Avanti Polar Lipids) were suspended at 1 mg/ml in buffer containing 25 mM Hepes pH 7.4, 100 mM NaCl and 1 mM dithiothreitol, and liposomes were formed by sonication followed by hydration. Purified proteins were incubated with liposomes for 15 min at room temperature and were centrifuged at 60000 rpm for 20 min at 25°C. Obtained supernatants and pellets were subject to SDS-PAGE. The ratio of the amount of protein binding to lipid to the amount of protein in supernatants was determined using the Bradford protein assays (Bio-Rad Protein Assays Kits #5000001).

Structure prediction of mBPI5
Since the crystal structure of mBPI5 was not available in PDB, to obtain the 3D structure of mBPI5, its sequence was submitted to online server, I-TASSER (Iterative Threading ASSEmbly Refinement), for sequence alignment and homology modeling. I-TASSER is an advanced protein homology algorithm, which uses multiple individual programs and steps as well as molecular dynamics to create protein structural models of submitted protein sequences [22,23]. Models of mBPI5 created by I-TASSER were structurally evaluated using Verify3D [24]. The best 3D model was selected according to rank based on the C-score, TM score and RMSD value. www.impactjournals.com/oncotarget

Statistical analysis
All data were presented as mean ± standard deviation (SD). All statistical analyses were executed by using SPSS 17.0 software (IBM, Chicago, IL, USA). A two-sided value of *p < 0.05, **p < 0.001 was considered to be statistically significant.


The Fertile Darwins: Epigenesis, Organicism, and the Problem of Inheritance

This essay explores the Darwinian imagination – an approach to exploring the basic ontology of nature that was shared by both Erasmus Darwin and his grandson, Charles Darwin. It focuses on Erasmus and Charles’s respective theories of generation, especially as laid out in Zoonomia (1794) and The Variation of Animals and Plants Under Domestication (1868), and their derivation from the longstanding opposition between theories of epigenesis and panspermia. Erasmus Darwin’s thinking, in particular, was torn between relatively closed and open conceptions of how organic structures assemble and reproduce. Charles, by contrast, worked hard to fashion his theory of pangenesis into a capacious model that would account for interactions of inheritance and development across all levels of physical and temporal scale. Yet beneath disagreements over the distribution of agency between matter and different sexual partners, both argued for an anti-holist, anti-organic ontology that consistently cleared space for more open, more contingent, and ultimately more ecological theories of nature. Ultimately this required a rejection both of the Romantic conception of organic life and Romantic aesthetics, in particular, the notion that the unity of aesthetic experience communicated something about the unity of natural systems. Finally, I will argue that Charles Darwin’s pangenic model – which relies upon the functional assemblage of elements derived from different lineages, on different timescales, and with distinct, non-overlapping, and incompletely integrated capacities – offers a unique way of understanding the problem of intellectual inheritance and the relation between the Darwins themselves. If most approaches to this problem have posited either a lineal genealogy of intellectual inheritance, or situated their work as reflections of a larger historical context, pangenesis can help to imagine intellectual history beyond our typically lineal or atmospheric models of influence. Biographical note Devin Griffiths is Associate Professor of English at the University of Southern California. His research examines the intersection of intellectual history, scientific literature, and the digital humanities, with emphasis on nineteenth-century British literature and science. At the center of his research is the question Romanticism and Victorianism on the Net #66-67 (Spring-Fall 2016). Special issue on ‘The Two Darwins’. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 2 of how literary form shapes our experience of time and natural systems. His work on this subject has appeared in ELH, Studies in English Literature, Victorian Studies, Nineteenth-Century Contexts, Literature Compass, and Book History. His first book, The Age of Analogy: Science and Literature Between the Darwins, published in 2016 by the Johns Hopkins University Press, examines how historical novels established a new relational understanding of history and furnished a new comparative method, shaping the disciplinary formations of both the life sciences and the humanities. It was shortlisted for the British Society of Literature and Science’s book prize, and was runner-up for the first book prize of the British Association for Romantic Studies, and will be issued in paperback in fall, 2019. He is at work on a new book project: “The Ecology of Form,” which examines how Darwinian philosophy offers alternative models for ecology and the study of literary form. Romanticism and Victorianism on the Net #66-67 (Spring-Fall 2016). Special issue on ‘The Two Darwins’. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 3 “Be careful the thought-seeds you plant in the garden of your mind, For each seed grows after its own kind.” Funkadelic, “Good Thoughts, Bad Thoughts” 1. Is there a “Darwinian imagination”?1 To date, much of the work on the relation between Erasmus Darwin and his more famous grandson has been focused on their scientific theories – especially how Erasmus’s theory of evolution might have influenced the theory of natural selection.2 A key challenge for studying the relation between the Darwins has been the wide difference in their reputations and in the afterlives of their most precious theories. Erasmus’s imaginative and poetic speculations about evolution were ridiculed by subsequent generations. Charles, by contrast, is now often recognized as the most important naturalist of the nineteenth century – as a paragon of Victorian scientific virtues rather than an epigone of his grandfather’s fanciful conjectures. Charles would later claim defensively that his grandfather’s thinking had not had “any effect” on his own.3 Even in my own work, I have argued that Charles succeeded where his grandfather failed, in shaping a coherent argument for evolution rooted in the differential and contingent narratives of natural selection.4 2. Even so, in asking whether there is such a thing as a “Darwinian imagination” – by which I mean a mode of speculative thought that bridges the various rhetorical, disciplinary, and historical divides between their writings – I pose the question of what their respective visions might share beyond the technical features of their specific theories of nature. In spite of his self-presentation as a sober Baconian empiricist (the qualifier “careful” is used more than thirty times in the first edition of On the Origin of Species) Charles Darwin’s notebooks and private papers show that, much like his grandfather, he was an extraordinarily ambitious and imaginative thinker. Privately Charles referred to these speculations as “castles in the air” – experiments in thought that attempted to frame out speculations that were often not amenable to experimental study.5 The hypothesis of natural selection was itself an example of such speculative thinking. 6 It has sometimes been easy to forget, especially given the powerful support provided by the “new synthesis” of genetics and Darwinian thought in the 20th century, just how tenuous the evidence for evolution seemed to be. A key object of the history and sociology of science in the last several decades has been to recognize the complex and uncertain place of such theories in their time and amid the debates that they spawned.7 The success of natural selection continues to make it hard Romanticism and Victorianism on the Net #66-67 (Spring-Fall 2016). Special issue on ‘The Two Darwins’. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 4 to see just how outré the theory seemed. And it is this outlandish ambition – recognized as the audacity of his various attempts to reimagine the natural world from the ground up – that continually brought Charles closer to his grandfather. 3. For this reason, the present essay argues that the common impulse of their imaginations is more clearly evinced by the basic presuppositions they shared about how the natural world works – what Louis Althusser termed the “spontaneous philosophies” of scientists – rather than in their developed theories.8 The theories of reproduction and generation produced by both Erasmus and Charles Darwin can be viewed as projections of their respective philosophical ontologies – their fundamental assumptions about the order of the natural world, its structures and operations. All writing has an ontology, but in scientific writing the ontology of nature is front and center and (to some degree of remove) is the central matter at hand. In nineteenth-century realist fiction, by way of contrast, the basic ontology of nature is not usually at stake.9 Science and nature writing (and here I include all the major works of both Erasmus and Charles) are centrally about a natural ontology, whether or not that ontology is brought to the fore as an object of explicit discussion. 4. The theories of reproduction furnished by Erasmus and Charles Darwin provide powerful portraits of those respective ontologies. In confronting the mechanisms of reproduction – the means by which features from the parents are transmitted, combined, and then grow exponentially into a new being through the assimilation of new matter – both Darwins were forced to articulate a basic ontology that answered key questions about the natural world: Is matter inert or vital? Where does organic organization come from? How does nonliving matter get converted into the stuff of life? In approaching these problems, the Darwins shared a basic and (to some degree) idiosyncratic belief that all life shares a common biological basis. In particular, they believed that plant and animal life not only share a common history, but share their basic mechanisms. Their commitment to a singular ontology of life is evident in all aspects of their respective careers and especially in their seemingly compulsive return to analogies drawn between plant and animal life, and between the vegetative and the human. This commitment to life’s shared basis was determinative in their theories of reproduction because it forced them to articulate those theories in a way that could identify sexual reproduction in animals – by way of the insemination of egg or womb – with the fertilization of plants by pollen. Moreover, this commitment to a shared-incommon life meant that their theories had to also address other, nonsexual forms of reproduction and growth, from reproduction by fission (simple division), and by budding (seen in plants, some monocellular organisms, and corals), to the regrowth of limbs in animals.10 In exploring the Romanticism and Victorianism on the Net #66-67 (Spring-Fall 2016). Special issue on ‘The Two Darwins’. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 5 sexual theories of reproduction offered by both Erasmus and Charles Darwin, it is therefore important to recognize that neither explore sexual reproduction as one generative mechanism among many, but rather, imagine a single differentiated system of assimilation and

2 of how literary form shapes our experience of time and natural systems. His work on this subject has appeared in ELH, Studies in English Literature, Victorian Studies, Nineteenth-Century Contexts, Literature Compass, and Book History. His first book, The Age of Analogy: Science and Literature Between the Darwins, published in 2016 by the Johns Hopkins University Press, examines how historical novels established a new relational understanding of history and furnished a new comparative method, shaping the disciplinary formations of both the life sciences and the humanities. It was shortlisted for the British Society of Literature and Science's book prize, and was runner-up for the first book prize of the British Association for Romantic Studies, and will be issued in paperback in fall, 2019. He is at work on a new book project: "The Ecology of Form," which examines how Darwinian philosophy offers alternative models for ecology and the study of literary form. 3 "Be careful the thought-seeds you plant in the garden of your mind, For each seed grows after its own kind." Funkadelic, "Good Thoughts, Bad Thoughts" 1. Is there a "Darwinian imagination"? 1 To date, much of the work on the relation between Erasmus Darwin and his more famous grandson has been focused on their scientific theories -especially how Erasmus's theory of evolution might have influenced the theory of natural selection. 2 A key challenge for studying the relation between the Darwins has been the wide difference in their reputations and in the afterlives of their most precious theories. Erasmus's imaginative and poetic speculations about evolution were ridiculed by subsequent generations. Charles, by contrast, is now often recognized as the most important naturalist of the nineteenth century -as a paragon of Victorian scientific virtues rather than an epigone of his grandfather's fanciful conjectures.
Charles would later claim defensively that his grandfather's thinking had not had "any effect" on his own. 3 Even in my own work, I have argued that Charles succeeded where his grandfather failed, in shaping a coherent argument for evolution rooted in the differential and contingent narratives of natural selection. 4 2. Even so, in asking whether there is such a thing as a "Darwinian imagination" -by which I mean a mode of speculative thought that bridges the various rhetorical, disciplinary, and historical divides between their writings -I pose the question of what their respective visions might share beyond the technical features of their specific theories of nature. In spite of his self-presentation as a sober Baconian empiricist (the qualifier "careful" is used more than thirty times in the first edition of On the Origin of Species) Charles Darwin's notebooks and private papers show that, much like his grandfather, he was an extraordinarily ambitious and imaginative thinker. Privately Charles referred to these speculations as "castles in the air" -experiments in thought that attempted to frame out speculations that were often not amenable to experimental study. 5 The hypothesis of natural selection was itself an example of such speculative thinking. 6 It has sometimes been easy to forget, especially given the powerful support provided by the "new synthesis" of genetics and Darwinian thought in the 20 th century, just how tenuous the evidence for evolution seemed to be. A key object of the history and sociology of science in the last several decades has been to recognize the complex and uncertain place of such theories in their time and amid the debates that they spawned. 7 The success of natural selection continues to make it hard 4 to see just how outré the theory seemed. And it is this outlandish ambition -recognized as the audacity of his various attempts to reimagine the natural world from the ground up -that continually brought Charles closer to his grandfather.
3. For this reason, the present essay argues that the common impulse of their imaginations is more clearly evinced by the basic presuppositions they shared about how the natural world workswhat Louis Althusser termed the "spontaneous philosophies" of scientists -rather than in their developed theories. 8 The theories of reproduction and generation produced by both Erasmus and Charles Darwin can be viewed as projections of their respective philosophical ontologies -their fundamental assumptions about the order of the natural world, its structures and operations. All writing has an ontology, but in scientific writing the ontology of nature is front and center and (to some degree of remove) is the central matter at hand. In nineteenth-century realist fiction, by way of contrast, the basic ontology of nature is not usually at stake. 9 Science and nature writing (and here I include all the major works of both Erasmus and Charles) are centrally about a natural ontology, whether or not that ontology is brought to the fore as an object of explicit discussion.

The theories of reproduction furnished by Erasmus and Charles Darwin provide powerful
portraits of those respective ontologies. In confronting the mechanisms of reproduction -the means by which features from the parents are transmitted, combined, and then grow exponentially into a new being through the assimilation of new matter -both Darwins were forced to articulate a basic ontology that answered key questions about the natural world: Is matter inert or vital?
Where does organic organization come from? How does nonliving matter get converted into the stuff of life? In approaching these problems, the Darwins shared a basic and (to some degree) idiosyncratic belief that all life shares a common biological basis. In particular, they believed that plant and animal life not only share a common history, but share their basic mechanisms. Their commitment to a singular ontology of life is evident in all aspects of their respective careers and especially in their seemingly compulsive return to analogies drawn between plant and animal life, and between the vegetative and the human. This commitment to life's shared basis was determinative in their theories of reproduction because it forced them to articulate those theories in a way that could identify sexual reproduction in animals -by way of the insemination of egg or womb -with the fertilization of plants by pollen. Moreover, this commitment to a shared-incommon life meant that their theories had to also address other, nonsexual forms of reproduction and growth, from reproduction by fission (simple division), and by budding (seen in plants, some monocellular organisms, and corals), to the regrowth of limbs in animals. 10 In exploring the Enlightenment vitalism with Linnaean taxonomy in far-reaching ways. And yet, as we will see, even as this theory emphasized a sensitive mechanism of centralized control, it opened up the developmental process to the intrusion of outside agencies -a possibility forestalled by alternative theories like preformation.
6. Charles's theory of "pangenesis," by contrast, completely rejected the notion of a central mechanism of organic control in favor of the relative autonomy of the body's structures and the "elective affinities" of their material components. Set out in his 1868 study, The Variation of Animals and Plants Under Domestication, the theory of pangenesis distributed the mechanisms of inheritance and growth even more widely across the body and its environment. Charles suggested that the sex cells do not organize inheritance and reproduction, but rather, that all cells and structures of the body spin off highly structured particles or "gemmules" that, in reproduction, gather within the sexual gametes and ultimately construct new offspring. Charles envisioned the body as less an organized whole, governed by centralized control and order, than a collective of relatively autonomous cellular agents (Darwin,. Romanticism and Victorianism on the Net #66-67 (Spring-Fall 2016). Special issue on 'The Two Darwins'. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 6 7. As I will explain, the two theorists staked out opposite positions on a much older debate between epigenesis (championed by Aristotle) and panspermia (attributed to Democritus). 11 Yet behind these distinctions between their theories of reproduction, I will argue, stood a more basic commitment to the autonomous and deeply relational nature of living systems and their capacity to self-assemble into larger complexes. Both Erasmus and Charles rejected a strongly programmatic nature in which God, the great designer, prescribed the features of living creatures.
Both imagined organisms as relatively autonomous and self-directed, not only in their actions, but in the mechanisms of reproduction and growth. This redistribution of agency and control, in turn, was a prerequisite for their respective thinking about evolution, not only because it furnished mechanisms by which differentiation and speciation might occur, but more importantly, because it rejected out of hand the notion of a fixed ontology, secured by a first cause, which would hold the development of living creatures within proscribed limits. Consequently, while an insistence on the relative autonomy of the organism would seem to reinforce our sense of its organic integrity, that is, our sense that any creature is a self-enclosed integration of both structure and function, we will see that both Erasmus and Charles understood that integrity as a more tenuous assemblage built up out the dynamic interaction and transformation of organic parts via interaction with the wider natural world. As Amanda Jo Goldstein describes it, such thinking encourages a "turn from self-sufficient integrity toward a proto-ecological notion of contingency and interrelation" (Goldstein,74 Garden and Zoonomia, was programmatic and deeply androcentric (a position radically revised in later works). 13 Arguing that a sensitive filament constituted the basis of the male contribution to reproduction, Erasmus posited that this filament decisively selects, absorbs, and accretes the 7 nutrition furnished by the womb, egg, or floral body, metabolically determining the ultimate development and structural features of the mature organism (Darwin, Zoonomia, 1794, 1:480-94). Theorizing that the living filament that seeds new life originated as an extension of the nervous system of the male parent, Erasmus Darwin argued that its specific configuration in higher organisms was partly determined by the mental state of the male partner at coitus, even going so far as to argue that it was this imaginative state (whether focused on the other partner, the self, or absent persons) that determined the sex of the child (1:514-9). In this way, Erasmus Darwin's reproductive theory recast procreation as an act of sexualized Romantic creation: the imaginative imposition of masculine form on feminine substance.
9. Yet this notion of masculine form is radically undermined in Zoonomia when it is placed within his wider framework for thinking about the ontology of nature. And a consideration of the longer history of thinking about ontogeny -the origin and development or organic being -can help explain why. 14 In arguing for epigenesis, Erasmus Darwin was intervening in a debate that was truly ancient. 15 Aristotle's On the Generation of Animals (referenced in Zoonomia) had extensively explored these two opposed schools of thought. 16 The first, the atomist school led by Democritus, argued for "panspermia" -the theory that young are generated by spermatic elements derived from all parts (pan) of the bodies of both parents. The alternative, advanced by Aristotle himself, held that the foetus developed epigenetically -that is, in reaction to the stimulation of the male spermatic material from "without" (epi). If, in the former view, offspring are formed by a union of elements derived from each parent in roughly equal measure, in the latter view, the female uterine material provided a kind of formless matter given shape and structure by the formative principle furnished by the male. inference, in which the male element serves as the controlling or formal cause that gives shape to the offspring through the mediating or material cause of the feminine tissue. In the former theory, agency is distributed; in the latter it is local and univocal.
11. This configuration was fundamentally reset in the Enlightenment, as new microscopic technologies made possible a series of experimental observations by William Harvey, Antonie van Leeuwenhoek,Nicolaas Hartsoeker,Nicolas Malebranche,and Charles Bonnet. 18 In studying the structure of the sperm and egg cells, these new "preformationists" became convinced that adults are an expansion of structures already contained, prepackaged and entire, in the germ line of the parent -though they disagreed whether it was the egg cell or the sperm that contained future generations. This new theory of preformation retooled causality by implying that all present life was predesigned and, in some sense, built into the original parents of any given species, however many generations ago. Preformation underlined a notion of the "first cause" consonant with Christian theology: whereas Aristotelian epigenesis located causality in the interaction between male and female reproductive elements, the new preformationists made cause both internal and anterior to the embryo.
12. Against this background, Erasmus Darwin presents an unsuccessful and, for that reason, fascinating attempt to return the problem of agency to an Aristotelian framework that saw causation as eminent within the world rather than ultimate. At the same time that Erasmus Darwin held that "the productions of nature are governed by general laws" (Zoonomia 1:485), his careful consideration of the implications of embryogenesis often pointed toward a notion of causality and agency that was far more distributed, even Democritean. For one, Erasmus held, with Linnaeus, that the hybridization of distinct species might have often led to successful new species, not just sterile "mules" (1:499, 514). Such full hybridization of species implied that the female parent must have significant influence over the structure of the offspring, an argument Erasmus implies in his critique of preformation (1:497).
13. Such considerations, Erasmus admitted, implied that the feminine "nutriment" was not simple matter but must consist of "ultimate particles" which formatively impact the development of the offspring. In later stages, Erasmus reasoned, the nervous system, through the action of a fully- formed digestive system, would be able to assert more control over its development, breaking down those "ultimate particles" into simpler units that could then be subsequently and selectively assimilated. But by that point, as shown by the production of hybrid species, the influence of the mother is equally determinative, scrambling Aristotle's strict distinction between formal cause and passive matter. If feminine "particles" help to determine the development and behavior of offspring, this means that the mother also contributes formally to the organization and action of the offspring. Far from passive matter, the uterus, egg, or floral body contributes important structural information to the young. Hence the androcentric impulse of generation fails to overwrite a more equitable contribution of both sexual partners to the formation of young. Such determinative and vital interactions are a general feature of matter, especially organic matter.
Zoonomia implies a basic ontology of life that the male filament is not able to overwrite, an ontology that admits the spontaneous volition of organic matter (whether derived from the female or male) to associate, interact, and assemble into more complex units. It is foundational to the "vitalist poetics" that, in Catherine Packham's analysis, underwrites his poetic and scientific writings (Packham,Chapter 5).
14. This tension between spermatic form and a more general and vital principle of assembly plays out at multiple points in Zoonomia, and is clearly registered wherever the problematic of causal agency inflects the syntax of his descriptions. Note the slippery agency in his extended later description of the relation between digestion and the apposition of material from the mother: In this process we must attend not only to the action of the living filament which receives a nutritive particle to its bosom, but also to the kind of particle, with respect to form, or size, or colour, or hardness, and secretion. Now as the first filament or entity cannot be furnished with the preparative organs above mentioned, the nutritive particles, which are at first to be received by it, are prepared by the mother; and deposited in the ovum ready for its reception. These nutritive particles must be supposed to differ in some respects, when thus prepared by different animals. They may differ in size, solidity, colour, and form; and yet may be sufficiently congenial to the living filament, to which they are applied, as to excite its activity by their stimulus, and its animal appetency to receive them, and to combine them with itself into organization. (1.512-3) The passive constructions are pointedly ambiguous: if the incomplete "first filament" cannot "be furnished" with digestive organs at the outset, the "nutritive particles" are, in fact successfully "prepared by the mother" and "deposited." Moreover, those particles then "excite" the "activity" of the filament, and even more tortuously, excite "its animal appetency … to combine them with itself into organization." So: the feminine particles excite the male filament to combine with the particles into organization. This syntactic construction completely deranges the Aristotelian 10 model, in which the male formative principle stimulates the potential form of the female into action. Here the potentiality of the male filament is activated and given determinative direction by the female elements and the structural (read formal) information they encode.
15. The distributive syntax creates an environment of shared agency, an environment that completely upends the connotative distinctions between actively formative male filament and receptive feminine elements, and cultivates a far more relational and distributed field of interaction. In such passages, Erasmus Darwin's theory of reproduction looks less like a theory of epigenesis than an early theorization of pangenesis. It implies an ontology in which the organism is not the product of a localized formative impulse, but rather, consists in the formative and contingent interaction of internal and exterior elements -an emergent ecology of interacting agents rather than a formal unity.
16. My point is that such moments mark the devolution of the sexual logic that Erasmus Darwin employed in his attempt to revive Aristotelian formalism as a model for the development of offspring. Such passages distribute agency and control, the distinction between formal impulse and matter, into a much wider, more sociable, and more distributed network of interaction, relation, and assembly. This is really of a piece with the cosmogony Erasmus laid out in his earlier writings. 19 These two trains of thought reflect two basic and deeply divided impulses in Erasmus Darwin's own thinking, a kind of formative tension between Aristotelian and Democritean schools. As I explain elsewhere, his Phytologia (1800) and Temple of Nature (1803) would eventually cashier the Aristotelian theory of epigenesis completely in favor of a model of elective affinity that offered a more distributed understanding of material, organic, and erotic agency. 20 In the third, "corrected" edition of Zoonomia, Erasmus makes the reversal indicated in Phytologia explicit: "I am now induced to believe, that the embryons of complicate animal and vegetable bodies are not formed from a single filament as above delivered; but that their structure commences in many parts at the same time" (Darwin, Zoonomia, 1801, 2:277-78  13 a microcosm -a little universe, formed of a host of self-propagating organisms, inconceivably minute and as numerous as the stars in heaven. (2:404) On first glance, Charles's ennobling vision of the organism as "a microcosm -a little universe, formed of a host of self-propagating organisms, inconceivably minute and as numerous as the stars in heaven" seems to reach for the sublime register his grandfather was famous for. Erasmus Darwin similarly imagined a deep coherence between the processes organizing microscopic, organismic, and cosmic life, but he constrained the ramifying complexity of that vision, closing out his discussion of generation with the argument that all development obeys a singular system of natural laws and a "Great First Cause" (known by tracing intermediate causes back to their source, "climbing up the links of these chains of being, till we ascend to the Great Source of all things" (1:533)). In place of a larger determining law or chain of causation, Charles underlines the unfathomable "complexity" of the organic system, and implicitly, the inscrutability of its basic principles.
21. Moreover, the intensely disorganizing power of Charles's vision is driven home by the startling analogy that precedes it. In imagining each creature as "a bed of mould full of seeds" -in other words, as a plot of soil in a garden -he looks beyond the notion of the organism as a closed environment completely. Instead, Charles argues, we must see our selves as physical ecologies: assemblages of organic and inorganic components, of organic nutrients and various cell lines, of processes of interaction, gestation, and propagation that operate on widely different scales. If some "seeds" (that is, cell lines and their traits) germinate soon, some reproduce on much longer time scales, while others pass away.
22. This vision of pangenesis is explicitly a system defined by contingent and chance events, from the chance that brings a given seed to maturity, to the chance that has brought these seeds together in the first place. Any living form is, in some sense, an accidental collection of the seeds or "gemmules" that happened to be around at the moment of reproduction, and while some of those gemmules then interact with others and their environment more or less harmoniously to form the offspring, many don't work at all, and many militate against its organic features. If Erasmus Darwin's Botanic Garden was wildly innovative and brought him widespread fame, particularly in the insistent analogies it drew between plant and human life, it did not propose that we view individual organisms as botanic gardens in themselves. 25 In his grandson's eyes, living forms, like any natural environment (from the garden plot to the seascape) are ecologies.

The audacity of Charles Darwin's ecological vision is clear when compared to two sources that
had a significant influence on his thinking about the relative autonomy of organic components -Claude Bernard's physiology and Richard Owen's major study of embryogenesis. The garden analogy employed in the passage above, for example, seems to have been suggested directly by a passage in Bernard's Tissus Vivants (1866). Bernard was a major proponent of cellular theory and advocated a focus on relative autonomy of the different tissues (or organs) within the body. 26 In order to tease out the implications for pangenesis, Charles summarizes the latter's account of organic autonomy: "Each organ, says Claude Bernard, has its proper life, its autonomy; it can develop and reproduce itself independently of the adjoining tissues" (2:368-9). Goethe seems also to have carefully digested. 28

(Charles Darwin, in his long introduction to Ernst
Krause's Erasmus Darwin (1879), credits Phytologia as originating "the view, now universally adopted, that a plant consists of a 'system of individuals', and not merely of a multiplication of similar organs." 29 ) 26. An extraordinary implication threads through these analogies: that the parts of both plants and animals, "like polypi or coral insects," might achieve an almost complete autonomy. Owen echoes this possibility (sounded by Goethe and -more quietly -Erasmus Darwin) and amplifies its range: The question, however, is whether the tree represents such a whole, or is equivalent to one of the organized individual members; whether the leaf and the stamen may not answer rather to different individuals of the 'body corporate,' than to the lung or the testis of any single individual. … Our first (and the common) notion of a tree and a reproductive trees (respectively, illustrations 1, 2, and 3 in figure 1). As Owen explains, in all three images, the first stage of reproduction is represented by the fertilization of an egg or ovule (b) by a sperm or pollen cell (a). The first generation (d) reproduces asexually -either by gemmation or parthenogenesis -into subsequent generations (e), before ultimately producing sexually mature bodies that enable the cycle to start again. Owen's point is that, read through an analogy drawn between different kinds of life, the distinction between generation, organism, and organ is radically unstable and perhaps even counterproductive. 18 as an index to the bounds Owen wishes to circumscribe around his anti-organic thought. If one implication of parthenogenesis is that all existing living creatures can find their place within a singular tree of life, Owen insists that man does not live within it.

III. Charles Darwin and the Anti-organic thought
29. Charles Darwin, by contrast, was happy to see all of such implications through to their most radical end. Charles Lyell once wrote to Darwin about his long personal struggle to finally "go the whole Ourang" and acknowledge the actual kinship between humanity and other forms of life. 30 For Lyell, the struggle was between his "reasoning towards transmutation" and his "sentiments and imagination," which balked. 31 Charles Darwin's "reason" and his "imagination," by contrast, were closely aligned: he often seems excited, even eager to "go the whole Ourang" -and whole polyp, the whole plant, even the whole "bed of mould." 30. The conflict that Lyell described between reason and the sentimental imagination strikingly anticipates Althusser's argument about the tension between scientific empiricism and ideology.
But my point is not that contemporary scientists were laboring under a "spontaneous philosophy" that Darwin heroically overcame, any more than it makes sense to claim that scientific discovery 31. Ever since groundbreaking work by Gillian Beer and George Levine, the field of science and literature has been focused on exploring how the literary imagination and its modes of writing contribute to science. 33 In my own work, I have looked at analogies, and the relational frameworks they create, as crucial to nineteenth-century science. The present article suggests that the spontaneous ontology of each scientist is also a pervasive and important constituent of the scientific imagination. #66-67 (Spring-Fall 2016). Special issue on 'The Two Darwins'. Guest-edited by Martin Priestman and Louise Lee (University of Roehampton) 19 32. Moreover, by insisting that such ontologies can be explored by means of the imaginative vision they support, I mean to raise the problem of the aesthetic criteria they entail, that is, the possibilities that are felt to "fit" within a given ontology, versus those that are felt to be discordant, jarring. Lyell's distinction between what his reasoning implied and what his "imagination and sentiments" were willing to accept points to precisely this sort of aesthetic judgment, a judgment marked by his negative reaction to the theory of natural selection as it was presented over the years. This form of judgment is marked also, I would suggest, in the moments above where Claude Bernard and Richard Owen pulled back from the most radical or jarring implications of their theories, the shift in rhetorical register interjected by Owen's "No!"

Romanticism and Victorianism on the Net
33. As Sianne Ngai observes, such "ugly feelings" demonstrate the role affect plays in mediating between ideology and experience, here, between "spontaneous philosophy" and empirical study (Ugly Feelings "Introduction"). Such feelings do not simply circumscribe; they help to gather in the imaginative reach and direct it toward a felt sense of the most appropriate whole. As Owen puts it, reflecting on his trees of life, "The analogy is beautifully and closely maintained throughout" (60). Ultimately, Owen's insistence that "The stomach of a man if detached would not live and reproduce its kind … nor would the lung manifest its individuality" underlines the organic boundaries of his imagination; he is unwilling to read human life out of the framework of a robust and mature organicism, and place it into contact with less highly "organized" life.
Owen, like Bernard and most of his contemporaries, was still fascinated by the closure of Romantic organicism, with its insistence (after Kant) that the unity of the organic whole and its parts evince a reciprocal relationship in which each is the means to the other's end. For Kant and others, the primary example of organicism is the plant. 34 Moreover, Kant insisted that the judgment of "unity" which we apply to the organism is rooted in a perception of purposiveness and applies to aesthetic perception in general. When we look to a work of art, Kant argued, our aesthetic appreciation is bound up in the work's purposiveness -the sense that whole and part are organized around some higher cause.
34. This powerful notion of an intrinsic relation between organic bodies and works of art has had a long afterlife in literary criticism. As Denise Gigante observes, Coleridge secured this conception of "multëity in unity" for Anglo-American literary criticism by arguing for its importance to our apprehension of both natural bodies and literary forms (Life,(22)(23). The aesthetic perception of such seemingly integrated and holistic unity, he insists, belies the basic incoherence and radical divergence in the various "scales of nature" -physical, temporal, organizational -that produces what we see. Rather than serving as an index to natural integrity, aesthetic perception is perceived as an impediment to grasping the complexity of natural systems.
Charles's consistent strategy in such passages is to read through the organic integrity and disassemble it, to insist on the basic disorganization of life. His quintessentially anti-organic move regarding forms, whether we talk of species, or social customs, or the beauty of an orchid, is to show that we make a mistake in seeing these forms as distinct, integral, and coherent structures. The organism and the ecology are both unstable constellations, captured within a shifting network of supportive alliances, shifting antagonisms, and the more pervasive condition of simply being thrown together.
38. Like his grandfather, Charles Darwin shows a startling propensity to think the hardest version of his thought -to push a given insight toward its most unsettling implications. If the theory of pangenesis fundamentally upset the organicism of contemporary scientific imagination of natural forms, this seems of a piece with a basic iconoclastic impulse in his writing. I have argued for the importance of disanalogy in George Eliot's writing as an epistemologically productive moment in which we are asked to recognize where a given model -a preconceived analogy -fails to account for what is encountered (Griffiths,The Age of Analogy,chap. 4). In a similar fashion I think that the Darwinian imagination -particularly in Charles's later writing -is predicated on dis-aestheticization, or better, a negative aesthetic or "ugly feeling" that rewards the effort to look behind the perception of aesthetic unity and perceive what it obscures. What is at stake is not a witheringly: the "enthusiastic advocacy of holism is not wholly derived from an objective contemplation of the facts of nature, but is at least partly motivated by an imagined future 'whole' to be realised in an ideal human society whose reflected glamour falls on less exalted wholes, illuminating with false light the image of the 'complex organism'" ("The Use and Abuse of Vegetational Concepts and Terms" 299). To truly appreciate Darwin's ecological vision, it would be necessary to come to terms with the continued "glamour" of the organism as a governing model for our conception of the earth and its systems.  Krause, in his brief study of Erasmus, accurately summarized this tendency: "We find the same indefatigable spirit of research, and almost the same biological tendency, as in his grandson; and we might, not without justice, assert that the latter has succeeded to an intellectual inheritance, and carried out a programme sketched forth and left behind by his grandfather" (Krause,Erasmus Darwin 132,emph. added). Such accounts, as Alexis Harley has recently observed, powerfully confuse notions of biological and intellectual relation. While inheritance is a particular problem for studying the Darwins, it has long been a central concern of the study of science and literature, particularly as that field developed from the older history of ideas in the tradition of A. O. Lovejoy and Marjorie Hope Nicolson (Rousseau,"Literature and Science,. Gillian Beer effectively reversed the problem of transmission with her influential call to examine the "twoway" traffic by which scientific theories and imaginative models are exchanged between both writers and scientists (Darwin's Plots 5). More recently, and with the advent of historicist and contextualizing approaches to the problem of cultural history, the "one culture" theorists have sought to locate both scientists and writers within larger determinative contexts (Levine, One

In
Culture "Introduction"). Instead of the integrity of the unit idea or specific strategies of literary representation, such thinking locates the integrity of influence at the level of the culture. While of seeds," the many intellectual seeds they helped germinate (only some of which they shared) were outnumbered by the many they did not: the seeds that remained dormant, the many more that never found their way into their respective plots of the intellectual landscape. Moreover, to truly see this vision through, we would have to recognize that these fertile constellations of thought are far less stable than the "unit ideas" envisioned by Lovejoy or even the "units of


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ITPR3 gene haplotype is associated with cervical squamous cell carcinoma risk in Taiwanese women

Host immunogenetic background plays an important role in human papillomavirus (HPV) infection and cervical cancer development. Inositol 1,4,5-triphosphate receptor type 3 (ITPR3) is essential for both immune activation and cancer pathogenesis. We aim to investigate if ITPR3 genetic polymorphisms are associated with the risk of cervical cancer in Taiwanese women. ITPR3 rs3748079 A/G and rs2229634 C/T polymorphisms were genotyped in a hospital-based study of 462 women with cervical squamous cell carcinoma (CSCC) and 921 age-matched healthy control women. The presence and genotypes of HPV in CSCC was determined. No significant association of individual ITPR3 variants were found among controls, CSCC, and HPV-16 positive CSCC. However, we found a significant association of haplotype AT between CSCC and controls (OR = 2.28, 95% CI 1.31–3.97, P = 2.83 × 10−3) and the OR increased further in CSCC patients infected with HPV-16 (OR = 2.89, 95% CI 1.55–5.37, P = 4.54 × 10−4). The linkage disequilibrium analysis demonstrated that ITPR3 association with CSCC was independent of HLA-DRB1 alleles. In conclusion, these findings suggest that AT haplotype in the ITPR3 gene may serve as a potential marker for genetic susceptibility to CSCC.

INTRODUCTION
Cervical cancer is the fourth most common cancer in women worldwide. In Taiwan, cervical cancer also poses a major public health concern, with nearly 2700 women were diagnosed with cervical cancer each year [1]. Infection with oncogenic human papillomavirus (HPV) is necessary for the development of cervical cancer [2]. However, the majority of women infected with HPV do not progress into cervical cancer, suggesting that other factors are required for the cancer to develop. A potential cofactor is the individual immune system, which may play a pivotal role in viral clearance and involve in the cervical cancer development.
The expression level of ITPR3 is associated with growth and aggressiveness of different types of tumors [9][10][11][12]. In addition, a number of studies have demonstrated that specific single nucleotide polymorphisms (SNPs) in the ITPR3 gene were associated with susceptibility to various immune-mediated diseases, such as type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, and Graves' disease [13][14][15].
Because of the importance of ITPR3 to the immunity and development of immune-associated diseases, it is reasonable to speculate that ITPR3 genetic variants might participate in the cancer pathogenesis. We therefore tested the hypothesis that specific ITPR3 SNPs are associated with cervical cancer risk with an association study of 462 cervical squamous cell carcinoma (CSCC) patients and 921 healthy controls.

RESULTS
A total of 84.7% detection rate for HPV DNA was found in 462 CSCC samples. The HPV type distributions were observed that HPV 16 was 63.5%, HPV 18 was 9.8%, and the remaining types were 26.7%.
The genotype and allele distributions of ITPR3 SNPs in cases and controls were shown in Tables 1 and 2. The genotype frequencies of all SNPs in the control group did not deviate significantly from Hardy-Weinberg equilibrium (P > 0.05). We did not find any significant differences in the genotype and allele frequencies for the 2 ITPR3 SNPs between CSCC patients and controls (Tables 1 and 2).
Based on the HPV-16 positivity stratification analysis, the synergistic effect of HPV-16 infection and P2RX7 polymorphisms on the risk of CSCC can be explored. The results showed that no significant difference of genotype and allele distributions was observed between women with HPV-16 positive CSCC and controls (Tables 1 and 2). Linkage disequilibrium (LD) analyses between the 2 SNPs revealed strong LDs in both controls (D′ = 0.87) and patients (D′ = 0.72).

DISCUSSION
In this study, we investigated the association of ITPR3 rs3748079 A/G and rs2229634 C/T SNPs and their haplotypes with the cervical cancer risk in Taiwanese women. The rationale for choosing these 2 variants is based on their potential functional implication in ITPR3 gene. The rs3748079 SNP, located in promoter region of ITPR3 gene, has been found to upregulate ITPR3 mRNA expression in cells having A allele [14]. Another variant, rs2229634, is a synonymous SNP situated in exon 20,   which might affect mRNA stability and splicing [16]. We found that, individually, rs3748079 and rs2229634 SNPs were not associated with CSCC susceptibility. However, the haplotype rs3748079 A-rs2229634 T conferred a risk of CSCC patients and the risk increased further in CSCC patients infected with HPV-16. The limitations of our study include a selection bias existing in the study of retrospective design and scarcity of screened SNPs. Therefore, a large-scale prospective study that investigates other SNPs of ITPR3 genes is needed to confirm our findings. Ca 2+ serves as a universal second messenger in virtually all cells. It plays a central role in regulating proliferation/activation and apoptosis of both immune and cancer cells [17,18]. When cells are challenged with diverse extracellular stimuli, phospholipase C pathway is activated and results in generation of IP3, which binds to IP3 receptors and releases Ca 2+ from the endoplasmic reticulum. IP3 receptors are a principal route of Ca 2+ flux and this makes them an excellent candidate for immune-and cancer-associated studies. An investigation performed by Khan et al. found that increased ITPR3 may be causally related to lymphocyte apoptosis [6]. Other reports showed that ITPR1 and ITPR3 are critical for T-cell activation because of their involvement in IL-2 and IFN-γ production [19,20]. In addition, a few studies found that overexpression of ITPR3 in breast and colon cancer cells decreased apoptosis, while knockdown of the receptor enhanced apoptosis [9,10]. Cancer cells also exploit various kinds of proto-oncogenes and tumorsuppressors to modulate the expression and function of IP3 receptors in their favor [21]. These findings strongly support a potential role for ITPR3 in carcinogenesis. However, the associations between individual ITPR3 gene polymorphism and CSCC risk have not been discovered in our study.
A haplotype is a set of DNA variations along a chromosome, which tends to be inherited together. A few studies have found that haplotypes may contribute to a phenotype through the combined effects of multiple individual SNPs on promoter activity or protein function [22,23]. The hypothesis that haplotypes are more important than SNPs has been supported by both simulated and empirical data. A simulation study reported by Akey et al. showed that haplotypes can significantly improve the power of an association test [24]. They also used published data from the hereditary hemochromatosis disease region to validate their findings. Another study conducted by Zhang et al. used coalescent simulations to infer haplotype frequencies and quantitatively assess the power in both case-control and case-parental control designs [25]. They found that haplotype-based analysis is always more powerful than marker-by-marker analysis. Fallin et al. applied expectation-maximization algorithm estimated haplotype frequencies to investigate associations between apolipoprotein E SNPs and Alzheimer's disease [26]. They showed that significant associations were obtained for haplotypes that were not identified using individual SNPs. These results clearly provide examples that haplotype analysis is more informative than association studies based on a single marker.
To the best of our knowledge, this study is the first to explore the association between ITPR3 gene haplotypes and cervical cancer. The AT haplotype was found to be associated with risk of women with CSCC and HPV-16 positive CSCC even though the rs3748079 A/G or rs2229634 C/T polymorphism alone was not. Thus, we speculate that the haplotype AT may   have a role in ITPR3 gene regulation and function and involve in a mechanism that determines the outcome of HPV infection and the subsequent development of CSCC. There are several possible explanations for these findings. The interaction of multiple SNPs within a haplotype may ultimately produce the biologic phenotype and become a powerful determinant of disease susceptibility. Another possibility is that the specific AT haplotype found in this study is in LD with the class II HLA loci.
Because ITPR3 locates at about 500kb centrometric to the class II HLA genes DR and DQ, which are found to associate with cervical cancer [27,28], it is important to clarify whether association between ITPR3 AT haplotype and CSCC is dependent on LD with class II HLA genes. The LD between ITPR3 and HLA-DRB1 alleles was analyzed in the subgroup of 333 CSCC patients and 241 controls using the PyPop software [29]. In this subgroup, we did not find any significant associations between HLA-DRB1 alleles and CSCC. Only weak LD was identified between ITPR3 and HLA-DRB1 alleles in the control subset (D′ = 0.31 and 0.34, respectively). These lines of evidence suggest that it is unlikely that the ITPR3 AT haplotype association with CSCC is dependent on HLA-DRB1 locus. However, we are unable to exclude the LD effect of HLA-DQB1 alleles or DRB1-DQB1 haplotypes on the association of ITPR3 AT haplotype with CSCC.
In summary, our study demonstrates that the AT haplotype in ITPR3 gene is markedly associated with CSCC risk in Taiwanese women. Additionally, women carrying the AT haplotype and harboring HPV-16 infection enhance susceptibility to CSCC. Further investigations are warranted to elucidate the specific role of ITPR3 molecule on the etiology of CSCC.

Study subjects
The study included 462 patients with CSCC (mean ± SD age at diagnosis: 53.2 ± 13.1 years) residing in northern Taiwan. The diagnosis of CSCC was confirmed by histological examinations of tissues from biopsies or resected specimens. The control group consisted of 921 sex-and age-matched healthy subjects (mean ± SD age at sampling: 52.4 ± 12.1 years) who were enrolled from women attending the routine Pap screening and with normal Pap smear and no previous history of cervical dysplasia. The cases and controls were all genetically unrelated Taiwanese women. Informed written consent was obtained from all participants for use of their surgical resections or cervical scrapings for the study. The study protocol conformed to the ethical guidelines of the 1964 Declaration of Helsinki and was approved by the Institutional Review Board of Mackay Memorial Hospital.

DNA extraction
Formalin-fixed, paraffin-embedded tissue blocks from CSCC patients were sectioned and dewaxed, and genomic DNA was then extracted using the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Genomic DNA of controls was extracted from cervical scrapings using Qiagen DNA extraction kit.

HPV detection and typing
The detection of HPV and genotyping on 462 cervical DNA samples were performed by polymerase

ITPR3 genotyping
ITPR3 rs3748079 A/G and rs2229634 C/T polymorphisms were genotyped. They were determined using the Pre-Developed TaqMan Allelic Discrimination Assay (Applied Biosystems, Foster City, CA). Briefly, polymerase chain reactions (PCR) were carried out in a 96well GeneAmp PCR System 9700 (Applied Biosystems) with mixes consisting of 10 ng of genomic DNA, 5 µl of TaqMan Universal PCR Master Mix, 0.5 µl of 20× Assay Mix, and ddH 2 O to a final volume of 10 µl. Thermal cycle conditions were as follows: denaturation at 95ºC for 10 min, followed by 40 cycles of denaturation at 92ºC for 15 sec, and annealing and extension at 60ºC for 1 min. After PCR, the TaqMan assay plates were transferred to the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) where the endpoint fluorescence intensity in each well of the plate was read. The allele specific fluorescence data from each plate were analyzed using the SDS v.1.1 software (Applied Biosystems) to automatically determine the genotype of each sample.

Statistical analysis
Genotype and allele frequencies of the ITPR3 rs3748079 A/G and rs2229634 C/T SNPs were determined by direct counting. The Hardy-Weinberg equilibrium was assessed for each SNP in both the control and case groups by chi-square analysis. Linkage analysis and the frequencies of ITPR3 haplotypes in controls and cases were estimated using the Haploview 4.2 program [32].
Statistical differences in the genotype, allele, and haplotype distributions between controls and cases were performed by chi-square test with Yates' correction or Fisher's exact test (when the number of subjects in a cell was <5). Odds ratios (OR) and 95% confidence intervals (CI) were also calculated. Bonferroni correction for multiple testing was applied by multiplying P value with the number of comparisons performed. P c values of less than 0.05 (2-tailed) were considered to be statistically significant.
Prior to the study, statistical power to detect effects of the ITPR3 SNPs on susceptibility to CSCC was calculated using the Quanto Ver. 1.1 software (Department of Preventive Medicine, University of Southern California, CA, USA). We designed the study to have a power >95% to determine a relative risk of 1.4 for the genotype of each SNP at a significance level of 0.05, with an estimated prevalence of CSCC of 360/100,000 [33].