Docosahexaenoic Acid Supplementation in Pregnancy Modulates Placental Cellular Signaling and Nutrient Transport Capacity in Obese Women.

Context: Maternal obesity in pregnancy has profound impacts on maternal metabolism and promotes placental nutrient transport, which may contribute to fetal overgrowth in these pregnancies. The fatty acid docosahexaenoic acid (DHA) has bioactive properties that may improve outcomes in obese pregnant women by modulating placental function. Objective: To determine the effects of DHA supplementation in obese pregnant women on maternal metabolism and placental function. Design: Pregnant women were supplemented with DHA or placebo. Maternal fasting blood was collected at 26 and 36 weeks' gestation, and placentas were collected at term. Setting: Academic health care institution. Subjects: Thirty-eight pregnant women with pregravid body mass index ≥30 kg/m2. Intervention: DHA (800 mg, algal oil) or placebo (corn/soy oil) daily from 26 weeks to term. Main Outcomes: DHA content of maternal erythrocyte and placental membranes, maternal fasting blood glucose, cytokines, metabolic hormones, and circulating lipids were determined. Insulin, mTOR, and inflammatory signaling were assessed in placental homogenates, and nutrient transport capacity was determined in isolated syncytiotrophoblast plasma membranes. Results: DHA supplementation increased erythrocyte (P < 0.0001) and placental membrane DHA levels (P < 0.0001) but did not influence maternal inflammatory status, insulin sensitivity, or lipids. DHA supplementation decreased placental inflammation, amino acid transporter expression, and activity (P < 0.01) and increased placental protein expression of fatty acid transporting protein 4 (P < 0.05). Conclusions: Maternal DHA supplementation in pregnancy decreases placental inflammation and differentially modulates placental nutrient transport capacity and may mitigate adverse effects of maternal obesity on placental function.

Matrix metalloproteinase-9-dependent mechanisms of reduced contractility and increased stiffness in the aging heart.

PURPOSE: Matrix metalloproteinases (MMPs) collectively degrade all extracellular matrix (ECM) proteins. Of the MMPs, MMP-9 has the strongest link to the development of cardiac dysfunction. Aging associates with increased MMP-9 expression in the left ventricle (LV) and reduced cardiac function. We investigated the effect of MMP-9 deletion on the cardiac ECM in aged animals. EXPERIMENTAL DESIGN: We used male and female middle-aged (10- to16-month old) and old (20- to 24-month old) wild-type (WT) and MMP-9 null mice (n = 6/genotype/age). LVs were decellularized to remove highly abundant mitochondrial proteins that could mask identification of relative lower abundant components, analyzed by shotgun proteomics, and proteins of interest validated by immunoblot. RESULTS: Elastin microfibril interface-located protein 1 (EMILIN-1) decreased with age in WT (p < 0.05), but not in MMP-9 null. EMILIN-1 promotes integrin-dependent cell adhesion and EMILIN-1 deficiency has been associated with vascular stiffening. Talin-2, a cytoskeletal protein, was elevated with age in WT (p < 0.05), and MMP-9 deficiency blunted this increase. Talin-2 is highly expressed in adult cardiac myocytes, transduces mechanical force to the ECM, and is activated by increases in substrate stiffness. Our results suggest that MMP-9 deletion may reduce age-related myocardial stiffness, which may explain improved cardiac function in MMP-9 null animals. CONCLUSIONS: We identified age-related changes in the cardiac proteome that are MMP-9 dependent, suggesting MMP-9 as a possible therapeutic target for the aging patient.

Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover.

Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly Fli-1. EWS-Fli-1 fusion accounts for 85% of cases. The growth and survival of Ewing sarcoma cells are critically dependent on EWS-Fli-1. A large body of evidence has established that EWS-Fli-1 functions as a DNA-binding transcription factor that regulates the expression of a number of genes important for cell proliferation and transformation. However, little is known about the biochemical properties of the EWS-Fli-1 protein. We undertook a series of proteomic analyses to dissect the EWS-Fli-1 interactome. Employing a proximity-dependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1 within a cell. CIMPR is a cargo that mediates the delivery of lysosomal hydrolases from the trans-Golgi network to the endosome, which are subsequently transferred to the lysosomes. Further molecular cell biological analyses uncovered a role for lysosomes in the turnover of the EWS-Fli-1 protein. We demonstrate that an mTORC1 active-site inhibitor, torin 1, which stimulates the TFEB-lysosome pathway, can induce the degradation of EWS-Fli-1, suggesting a potential therapeutic approach to target EWS-Fli-1 for degradation.

Proteomic analysis of the left ventricle post-myocardial infarction to identify in vivo candidate matrix metalloproteinase substrates.

Left ventricular remodeling post-myocardial infarction (MI) involves a multitude of mechanisms that regulate the repair response. Matrix metalloproteinases (MMPs) are a major family of proteolytic enzymes that coordinate extracellular matrix turnover. MMP-7 or MMP-9 deletion attenuate adverse remodeling post-MI, but the mechanisms have not been fully clarified. Both MMP-7 and MMP-9 have a large number of known in vitro substrates, but in vivo substrates for these two MMPs in the myocardial infarction setting are incompletely identified. Advances in proteomic techniques have enabled comprehensive profiling of protein expression in cells and tissue. In this chapter, we describe a protocol for the proteomic analysis of in vivo candidate MMP substrates in the post-MI left ventricle using two-dimensional electrophoresis, liquid chromatography coupled with tandem mass spectrometry, and immunoblotting.

Texas 3-step decellularization protocol: looking at the cardiac extracellular matrix.

The extracellular matrix (ECM) is a critical tissue component, providing structural support as well as important regulatory signaling cues to govern cellular growth, metabolism, and differentiation. The study of ECM proteins, however, is hampered by the low solubility of ECM components in common solubilizing reagents. ECM proteins are often not detected during proteomics analyses using unbiased approaches due to solubility issues and relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. Decellularization has become a common technique for ECM protein-enrichment and is frequently used in engineering studies. Solubilizing the ECM after decellularization for further proteomic examination has not been previously explored in depth. In this study, we describe testing of a series of protocols that enabled us to develop a novel optimized strategy for the enrichment and solubilization of ECM components. Following tissue decellularization, we use acid extraction and enzymatic deglycosylation to facilitate re-solubilization. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. These fractions, developed in mass spectrometry-compatible buffers, are amenable to proteomics analysis. The developed protocol allows identification (by mass spectrometry) and quantification (by mass spectrometry or immunoblotting) of ECM components in tissue samples. BIOLOGICAL SIGNIFICANCE: The study of extracellular matrix (ECM) proteins in pathological and non-pathological conditions is often hampered by the low solubility of ECM components in common solubilizing reagents. Additionally, ECM proteins are often not detected during global proteomic analyses due to their relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. In this manuscript we describe testing of a series of protocols that enabled us to develop a final novel optimized strategy for the enrichment and solubilization of ECM components. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. By analysis of each independent fraction, differences in protein levels can be detected that in normal conditions would be masked. These fractions are amenable to mass spectrometry analysis to identify and quantify ECM components in tissue samples. The manuscript places a strong emphasis on the immediate practical relevance of the method, particularly when using mass spectrometry approaches; additionally, the optimized method was validated and compared to other methodologies described in the literature.

Profiling the anaerobic response of C. elegans using GC-MS.

The nematode Caenorhabditis elegans is a model organism that has seen extensive use over the last four decades in multiple areas of investigation. In this study we explore the response of the nematode Caenorhabditis elegans to acute anoxia using gas-chromatography mass-spectrometry (GC-MS). We focus on the readily-accessible worm exometabolome to show that C. elegans are mixed acid fermenters that utilize several metabolic pathways in unconventional ways to remove reducing equivalents - including partial reversal of branched-chain amino acid catabolism and a potentially novel use of the glyoxylate pathway. In doing so, we provide detailed methods for the collection and analysis of excreted metabolites that, with minimal adjustment, should be applicable to many other species. We also describe a procedure for collecting highly volatile compounds from C. elegans. We are distributing our mass spectral library in an effort to facilitate wider use of metabolomics.

Wnt antagonist SFRP1 functions as a secreted mediator of senescence.

Cellular senescence has emerged as a critical tumor suppressive mechanism in recent years, but relatively little is known about how senescence occurs. Here, we report that secreted Frizzled-related protein 1 (SFRP1), a secreted antagonist of Wnt signaling, is oversecreted upon cellular senescence caused by DNA damage or oxidative stress. SFRP1 is necessary for stress-induced senescence caused by these factors and is sufficient for the induction of senescence phenotypes. We present evidence suggesting that SFRP1 functions as a secreted mediator of senescence through inhibition of Wnt signaling and activation of the retinoblastoma (Rb) pathway and that cancer-associated SFRP1 mutants are defective for senescence induction.

Plasminogen activator inhibitor 1--insulin-like growth factor binding protein 3 cascade regulates stress-induced senescence.

Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescence-inducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.

Plasma fractionation enriches post-myocardial infarction samples prior to proteomics analysis.

Following myocardial infarction (MI), matrix metalloproteinase-9 (MMP-9) levels increase, and MMP-9 deletion improves post-MI remodeling of the left ventricle (LV). We provide here a technical report on plasma-analysis from wild type (WT) and MMP-9 null mice using fractionation and mass-spectrometry-based proteomics. MI was induced by coronary artery ligation in male WT and MMP-9 null mice (4-8 months old; n = 3/genotype). Plasma was collected on days 0 (pre-) and 1 post-MI. Plasma proteins were fractionated and proteins in the lowest (fraction 1) and highest (fraction 12) molecular weight fractions were separated by 1-D SDS-PAGE, digested in-gel with trypsin and analyzed by HPLC-ESI-MS/MS on an Orbitrap Velos. We tried five different fractionation protocols, before reaching an optimized protocol that allowed us to identify over 100 proteins. Serum amyloid A substantially increased post-MI in both genotypes, while alpha-2 macroglobulin increased only in the null samples. In fraction 12, extracellular matrix proteins were observed only post-MI. Interestingly, fibronectin-1, a substrate of MMP-9, was identified at both day 0 and day 1 post-MI in the MMP-9 null mice but was only identified post-MI in the WT mice. In conclusion, plasma fractionation offers an improved depletion-free method to evaluate plasma changes following MI.

Characterization of lytic Pseudomonas aeruginosa bacteriophages via biological properties and genomic sequences.

Pseudomonas aeruginosa is an important cause of infections, especially in patients with immunodeficiency or diabetes. Antibiotics are effective in preventing morbidity and mortality from Pseudomonas infection, but because of spreading multidrug-resistant bacterial strains, bacteriophages are being explored as an alternative therapy. Two newly purified broad host range Pseudomonas phages, named vB_Pae-Kakheti25 and vB_Pae-TbilisiM32, were characterized as candidates for use in phage therapy. Morphology, host range, growth properties, thermal stability, serology, genomic sequence, and virion composition are reported. When phages are used as bactericides, they are used in mixtures to overcome the development of resistance in the targeted bacterial population. These two phages are representative of diverse siphoviral and podoviral phage families, respectively, and hence have unrelated mechanisms of infection and no cross-antigenicity. Composing bactericidal phage mixtures with members of different phage families may decrease the incidence of developing resistance through a common mechanism.

The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the ß domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.

Proteomic dissection of the von Hippel-Lindau (VHL) interactome.

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex containing elongin B, elongin C, cullin 2, and Rbx1, which acts as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the alpha subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in the maintenance of primary cilium, extracellular matrix formation, and tumor suppression. We undertook a series of proteomic analyses to gain a comprehensive picture of the VHL-interacting proteins. We found that the ARF tumor suppressor interacts with VHL30, a longer VHL isoform, but not with VHL19, a shorter VHL isoform. ARF was found to release VHL30 from the E3 ligase complex, promoting the binding of VHL30 to a protein arginine methyltransferase, PRMT3. Our analysis of the VHL19 interactome also uncovered that VHL19 displays an affinity to collagens and their biosynthesis enzymes.

Quantitative proteomic identification of the BRCA1 ubiquitination substrates.

Mutation of the BRCA1 tumor suppressor gene predisposes women to hereditary breast and ovarian cancers. BRCA1 forms a heterodimer with BARD1. The BRCA1/BARD1 heterodimer has ubiquitin ligase activity, considered to play crucial roles in tumor suppression and DNA damage response. Nevertheless, relevant BRCA1 substrates are poorly defined. We have developed a new approach to systematically identify the substrates of ubiquitin ligases by identifying proteins that display an enhanced incorporation of His-tagged ubiquitin upon ligase coexpression; using this method, we identified several candidate substrates for BRCA1. These include scaffold attachment factor B2 (SAFB2) and Tel2 as well as BARD1. BRCA1 was found to enhance SAFB protein expression and induce Tel2 nuclear translocation. Identification of the ubiquitination substrates has been a major obstacle to understanding the functions of ubiquitin ligases. The quantitative proteomics approach we devised for the identification of BRCA1 substrates will facilitate the identification of ubiquitin ligase-substrate pairs.

Peroxynitrite-induced nitrative and oxidative modifications alter tau filament formation.

Tau undergoes numerous posttranslational modifications during the progression of Alzheimer's disease (AD). Some of these changes accelerate tau aggregation, while others are inhibitory. AD-associated inflammation is thought to create oxygen and nitrogen radicals such as peroxynitrite (PN). In vitro, PN can nitrate many proteins, including tau. We have previously demonstrated that tau's ability to form filaments is profoundly affected by treatment with PN and have attributed this inhibition to tyrosine nitration. However, PN is highly reactive and unstable leading to oxidative amino acid modifications through its free radical byproducts. To test whether PN can modify other amino acids in tau via oxidative modifications, a mutant form of the tau protein lacking all tyrosines (5XY → F) was constructed. 5XY → F tau readily forms filaments; however, like wild-type tau the extent of polymerization was greatly reduced following PN treatment. Since 5XY → F tau cannot be nitrated, it was clear that nonnitrative modifications are generated by PN treatment and that these modifications change tau filament formation. Mass spectrometry was used to identify these oxidative alterations in wild-type tau and 5XY → F tau. PN-treated wild-type tau and 5XY → F tau consistently displayed lysine formylation throughout tau in a nonsequence-specific distribution. Lysine formylation likely results from reactive free radical exposure caused by PN treatment. Therefore, our results indicate that PN treatment of proteins in vitro cannot be used to study protein nitration as it likely induces numerous other random oxidative modifications clouding the interpretations of any functional consequences of tyrosine nitration.

Chemical modification of the Rieske protein from Thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2FE-2S] cluster.

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc(1) complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.

Proteomic analysis reveals late exercise effects on cardiac remodeling following myocardial infarction.

Exercise has been shown to improve function of the left ventricle (LV) following myocardial infarction (MI). The mechanisms to explain this benefit have not been fully delineated, but may involve improved mechanics resulting in unloading effects and increased endothelial nitric oxide synthase levels [1,2]. Accordingly, the goal of this study was to determine how the LV infarct proteome is altered by a post-MI exercise regimen. Sprague-Dawley rats underwent ligation of the left descending coronary artery to induce MI. Exercise training was initiated four weeks post-MI and continued for 8 weeks in n=12 rats. Compared with the sedentary MI group (n=10), the infarct region of rats receiving exercise showed 20 protein spots with altered intensities in two-dimensional gels (15 increased and 5 decreased; p<0.05). Of 52 proteins identified in 20 spots, decreased levels of voltage-dependent anion-selective channel 2 and increased levels of glutathione perioxidase and manganese superoxide were confirmed by immunoblotting. Cardiac function was preserved in rats receiving exercise training, and the beneficial effect was linked with changes in these 3 proteins. In conclusion, our results suggest that post-MI exercise training increases anti-oxidant levels and decreases ion channel levels, which may explain, in part, the improved cardiac function seen with exercise.

Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction.

Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48+/-3 in wt and 45+/-3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.

Proteome of the large Pseudomonas myovirus 201 phi 2-1: delineation of proteolytically processed virion proteins.

Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.

In vivo matrix metalloproteinase-7 substrates identified in the left ventricle post-myocardial infarction using proteomics.

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

DNA packaging-associated hyper-capsid expansion of bacteriophage t3.

Evidence that in vivo bacteriophage T3 DNA packaging includes capsid hyper-expansion that is triggered by lengthening of incompletely packaged DNA (ipDNA) is presented here. This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNA-containing capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids are based on two-dimensional, non-denaturing agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDS-PAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The following hypotheses are presented: (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.