Efficacy of free gingival grafting to augment keratinized mucosa around dental implants in posterior regions after restorative procedures: A retrospective clinical study.

STATEMENT OF PROBLEM: Data on the shrinkage of free gingival grafts (FGGs) vary. Most studies have analyzed grafts in nonmolar sites because of measurement limitations and have addressed the changes in grafts and keratinized mucosa width (KMW) only in the early healing phase. PURPOSE: The purpose of this retrospective clinical study was to assess the dimensional changes of an FGG in the posterior regions and their influencing factors, with the aim of obtaining sufficient and stable KMW after restoration. MATERIAL AND METHODS: A total of 77 implants in 40 participants who had undergone an FGG surgery were recruited. Graft sizes during surgery and the surface areas of keratinized mucosa at the follow-up visit after restorations were compared by digital analysis and verified by clinical measurements and photographs. The association between shrinkage and the graft sizes, implant location, and sex and age of the participants was evaluated. The influence of the shrinkage of FGG on the KMW after restoration was analyzed by multivariable linear regression with generalized estimating equation (GEE) models. RESULTS: The mean ±standard deviation shrinkage of FGG around implants in the posterior regions was 24.76 ±14.77%, and the mean ±standard deviation KMW was 4.16 ±1.77 mm at the follow-up visit. Larger grafts had a statistically higher shrinkage ratio (P<.001). No statistically significant difference was found regarding the effect of implant location, sex, and age on the shrinkage of FGG and final KMW (P>.05). The mean ±standard deviation follow-up period after restoration was 12.45 ±7.73 months CONCLUSIONS: Free gingival grafting was found to be a predictable treatment approach for augmentation of KMW around implants in the posterior region after the fabrication of prostheses as long as grafts of sufficient size were placed. Stable outcomes were shown in the study participants in the follow-up period of up to 3 years.

A Socket Seal Technique With the Use of Autologous Dental Roots for Socket Seal: A Case Series.

The literature identified variations in socket seal surgery, each with limitations. This case series aimed to observe the outcome of using autologous dental root (ADR) for socket sealing on socket preservation (SP). A total of 9 patients with 15 extraction sockets were documented. After flapless extraction, the xenograft or alloplastic grafts were placed in the sockets. Autologous dental roots were prepared extraorally and applied to seal the socket entrance. All SP sites healed uneventfully. Cone-beam computed tomography (CBCT) scan was performed after 4-6 months of healing to evaluate ridge dimensions. The preserved alveolar ridge profiles were verified on CBCT scans and during implant surgery. Implants were placed successfully with a reduced need for guided bone regeneration. Histological biopsy specimens were examined in 3 cases. The histological examination demonstrated vital bone formation and osseointegration of graft particles. All patients completed the final restorations and were monitored for 15.56 ± 9.08 months after functional loading. The favorable clinical outcomes support the use of ADR for SP procedures. It was not only accepted to patients but also easy to perform with low complication rates. The ADR technique is thus a feasible method for socket seal surgery.

Characteristics of the convergent angles of tapered implants based on a premolar root model.

STATEMENT OF PROBLEM: Developing tapered implants with the most appropriate angular characteristics requires an improved analysis of the anatomy of premolar roots. PURPOSE: The purpose of this observational study was to analyze the 3D anatomy of premolar roots by determining the tapered slope and convergent angle (TS/CA), to transform the TS/CA patterns into those in which the tapered implants mimic natural tooth roots, and to provide TS/CA references for future investigations. MATERIAL AND METHODS: A total of 73 human single-rooted premolars were surveyed and analyzed by microcomputed tomography and an associated software program. The 3D root surface area (RSA), the radius/diameter (R/D) at the planned first to tenth millimeter levels apical to the cementoenamel junction (CEJ), and the TS/CA at corresponding levels were calculated. The results were statistically analyzed by using an independent samples t test to assess the general differences of tested parameters between maxillary and mandibular premolars. A paired t test was used to examine the significant intragroup TS/CA differences between sequential coronoapical levels. One-way ANOVA was applied to study the general significance of developmental patterns in maxillary and/or mandibular groups. Two-way ANOVA was used to inspect the TS/CA significance at various measurements coronoapically between the maxillary and mandibular premolars (α=.05). RESULTS: Generally, the RSA, root length, R/D, and TS/CA parameters examined for the maxillary premolar roots differed significantly from those for the mandibular roots at the evaluated levels (P<.05). According to the measurements, the maxillary premolar roots generally exhibited nonsignificant RSA and R/D reduction patterns, with a decreasing angle of TS=13.44 degrees and CA=24.53 degrees coronoapically. However, mandibular premolar roots exhibited a significant reduction pattern, with TS=11.25 degrees and CA=21.06 degrees coronoapically according to both individual and general evaluations. CONCLUSIONS: Based on the developmental patterns of the evaluated TSs/CAs, tapered implants imitating premolar root anatomy should have a conical rather than a cylindrical shape, and the R/D of these models should be reduced to half at the apical third. However, further studies are warranted to identify more TS/CA characteristics related to the tapered implants, including the TSs/CAs of other tooth types.

Morphology of Peri-Implant Tissues Around Permanent Prostheses With Various Emergence Angles Following Free Gingival Grafting.

PURPOSE: To analyze the tissue morphology around implant-supported prostheses by digital technology and to evaluate the effect of prosthetic contours on the changes in tissues following the free gingiva graft procedure. MATERIAL AND METHODS: A total of 53 implants in 32 patients receiving free gingiva grafts were selected. These had previously presented insufficient keratinized mucosa width (KMW). At the follow-up visits (mean: 16.66 ± 9.97 months), the implant position and tissue condition were documented with an oral scanner. Vertical soft tissue thickness (VT), measured from the implant-abutment connection to the marginal tissues, and horizontal soft tissue thickness (HT), at the level of the platform, were calculated. The VT, HT, and emergence angle (EA) of prostheses were assessed by 3Shape analyzing software. The final KMW was measured by clinical assessment. Marginal bone loss (MBL) was calculated in the follow-up bitewing radiographs. RESULTS: The mean VT in the study was 2.65 ± 0.75 mm at the mid-buccal sites, 3.74 ± 1.22 mm at the mesial, 3.16 ± 1.08 mm at the distal, and 2.53 ± 0.92 at the mid-lingual aspects. The mid-buccal HT was 1.45 ± 0.53 mm while the mid-lingual was 1.05 ± 0.43 mm (p = 0.008). Interestingly, prostheses with mid-buccal EA > $\; > \;$ 30° exhibited slightly lower VT, but higher HT, than the ones with EA ≤ $\; \le \;$ 30°. Prostheses with proximal EA > 30° displayed slightly more MBL, compared to prostheses with EA ≤ $\; \le \;$ 30°. The mean KMW was 4.08 ± 1.10 mm. CONCLUSIONS: Free gingival grafting is a predictable treatment approach to augmenting soft tissue 3-dimensionally. Prostheses with EA ≤ $\; \le \;$ 30° were preferable for preserving the maximal VT and maintaining crestal bone stability.

The correspondence of 3D supporting bone loss and crown-to-root ratio to periodontitis classification.

AIM: Assessing the application of three-dimensional clinical attachment loss (3D-CAL), 3D supporting bone loss (3D-SBL), supracrestal tissue attachment (STA), and crown-to-root ratio (CRR) in evaluating the 2017 periodontitis classification. MATERIALS AND METHODS: We analysed ninety single-rooted human premolars with micro-computed tomography. The amount of 3D-SBL, linear radiographic bone loss (RBL), and CRR corresponding to various periodontitis stages as well as the statistical significance was investigated. RESULTS: From a 3D perspective, the premolars with a 21% of 3D-SBL at 2.0 mm coronal root length (RL) and 15% RBL corresponded to the periodontitis stage I. Premolars with a 44% of 3D-SBL at coronal 4.2-4.4 mm RL and 33% RBL accorded with the periodontitis stage II. Excluding the consideration of STA, CRR = 5:6 and 4:3 were associated with the levels at 15% and 33% RBL, respectively. CONCLUSIONS: A greater percentage of 3D-CAL than that of 2D-CAL is significant at evaluated levels. It is feasible to correlate the 3D-SBL, 3D-CAL, and STA parameters to evaluate the stages of periodontitis severity. However, the current use of RBL and CAL as applied for staging in the 2017 classification might be inconsistent with the evaluated premolar roots length, when STA dimensions are considered.

Fe-vacancy order and superconductivity in tetragonal ß-Fe1-xSe.

Several superconducting transition temperatures in the range of 30-46 K were reported in the recently discovered intercalated FeSe system (A1-xFe2-ySe2, A = K, Rb, Cs, Tl). Although the superconducting phases were not yet conclusively decided, more than one magnetic phase with particular orders of iron vacancy and/or potassium vacancy were identified, and some were argued to be the parent phase. Here we show the discovery of the presence and ordering of iron vacancy in nonintercalated FeSe (PbO-type tetragonal ß-Fe1-xSe). Three types of iron-vacancy order were found through analytical electron microscopy, and one was identified to be nonsuperconducting and magnetic at low temperature. This discovery suggests that the rich-phases found in A1-xFe2-ySe2 are not exclusive in Fe-Se and related superconductors. In addition, the magnetic ß-Fe1-xSe phases with particular iron-vacancy orders are more likely to be the parent phase of the FeSe superconducting system instead of the previously assigned ß-Fe1+δTe.

CCT- and CRI-tuning of white light-emitting diodes using three-dimensional non-close-packed colloidal photonic crystals with photonic stop-bands.

This study exhibited the correlated color temperature (CCT)- and color-rendering index (CRI)-tuning behavior of light emission from white light-emitting diodes (WLEDs) using three-dimensional non-close-packed (3D NCP) colloidal photonic crystals (CPhCs). The CCT of approximately 5300 K (characteristic of cold WLEDs) of white light propagated through the NCP CPhCs dropped to 3000 K (characteristic of warm WLEDs) because of the photonic stop-bands based on the photonic band structures of NCP CPhCs. This study successfully developed a novel technique that introduces lower-cost CCT- and CRI-tuning cold WLEDs with a CRI of over 90 that of warm WLEDs by using 3D NCP CPhCs.

Seaward Migration and Larval Release Coincide with Lunar and Light-dark Cycles in Supratidal Land Crabs Cardisoma carnifex and Epigrapsus notatus.

Herein we investigated the synchronous breeding migration and larval release of ovigerous females in two dominant supratidal land crabs Cardisoma carnifex and Epigrapsus notatus in the mixed semidiurnal tidal regime in Taijiang National Park (Tainan, Taiwan). We mainly focused on the monthly and daily rhythms during the breeding season of migration and larval release for these two crabs. We also sought to understand what the main environmental cues were for these monthly and diel rhythms. Both lunar and tidal amplitude cycles are potential proximate causes for the monthly lunar/semilunar reproductive rhythm in crabs. Likewise, either the 24-hour (diel) light cycle or tidal cycle can act as the proximate cause for diel reproduction rhythm, and we investigated which one was the main factor that entrains the diel rhythm for these two species. We found that the season of migration and larval release in C. carnifex occured mainly between June and September during the rainy season while those of E. notatus occurred mainly between September and October, near the end of the rainy season. Regarding the rhythm of migration and larval release in monthly time scale, C. carnifex exhibited a semilunar rhythm following the syzygies and E. notatus exhibited a lunar rhythm following the full moon. However, these rhythms did not occur with the maximum amplitude nocturnal and diurnal high tides. This implies that the lunar cycle is a more important environmental cue than the tidal amplitude in the entrainment of the synchronous monthly breeding rhythm for these two species. This pattern is different from other intertidal crabs, most of which use the tidal amplitude cycle as the main environmental cue for larval release. In addition to Chiromantes haematocheir (a supratidal crab), our study provided two more species that live in the supratidal zone time their reproduction with respect to the lunar light cycle and independent of the tide amplitude cycles. For the diel rhythm, both species migrated to the shore and released larvae in the first half of the night during the flood tide. This suggests that the diel light cycle is a dominant cue for the determination of larval release timing for these two species. Larval release does not track the high slack tides, since larvae are only released during the first half of the night and these high slack tides occur only after midnight (0000-0600H) during the days of larval release for these two crab species.

Convergent angles of a tapered implant referred from the root profile of premolars.

Background/purpose: Limited studies have discussed the convergent profiles regarding tapered implants based on biological considerations. This study analyzed the convergent angles (CAs) of premolar roots and imitated a tapered implant according to the anatomy of tooth roots. Materials and methods: A total of 60 single-rooted premolars were explored by micro-computed tomography. Every individual root was divided into 10 segments corono-apically, and the roots' buccolingual (BL) and mesiodistal (MD) CAs were measured by sections. To mimic a dental implant, the irregular shape of examined root cross-sections was transformed into a circular shape with equal areas. A biomimetic dental implant (BDI) was reconstructed and its CAs were compared with those of the natural roots' BL and MD at the examined levels and overall estimation. Results: In general, the maxillary and mandibular premolars demonstrated comparable CA patterns. However, significantly different CA patterns of BL, MD, and BDI were developed for both the maxillary and mandibular roots at the examined levels. The BL's CAs were greater than those CAs measured from the BDI and MD aspects, particularly for the sections at the middle and apical thirds of the roots. For overall CAs, the BDI's CAs were comparable with the average CAs of the BL and MD for both premolar groups. Conclusion: Instead of a cylindrical configuration, the BDI prototype demonstrated a tapered model with a continuous slope. The average CA of BDI was 14°-24°, serving as a biological reference for future tapered implant design and research.

The developing patterns of calibrated implant stability quotients of posterior implants.

BACKGROUND/PURPOSE: Many surgical protocols were modified to improve implant stability. However, the conclusions of applying osteotome condensation technique could enhance implant stability were controversial. The evaluated implant stability quotients (ISQ) were calibrated to differentiate the implant stability improvement that applied by varied surgical techniques and the bone quality at recipient sites. Therefore, this study examine the developing patterns of calibrated ISQ values induced by osteotome bone condensation and conventional drilling technique at the posterior ridges. MATERIALS AND METHODS: The ISQ values of 4.1/4.8-mm diameter implants were calibrated by 3.3-mm diameter implants (ISQb). Osteotome condensation technique was applied on the sites with ISQb ≤ 65 while those with ISQb > 65 were treated with conventional drilling technique. The implant ISQ values at Week 0, 1, 2, 3, 4, 6, 8, 10, 12 were recorded. The detected and calibrated ISQ values were statistically analyzed. RESULTS: Maxillary 14 implants and mandibular 16 implants using osteotome technique, maxillary 15 implants and mandibular 16 implants with conventional drilling technique were studied. Both techniques showed a generally similar ISQ developing pattern at both arches. Without calibration, significantly less ISQ values were noted for the osteotome technique of posterior maxilla at initial four weeks; subsequently, both techniques presented a comparable ISQ developing pattern. Osteotome technique demonstrated a greater ISQ increase after calibration on both arches (p < 0.05). All implants reached an ISQ stability plateau between Week 8 and 10. CONCLUSION: With calibration, osteotome condensation technique could enhance greater primary and secondary implant stability for both arches.

Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches.

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.

The fusion peptide domain is the primary membrane-inserted region and enhances membrane interaction of the ectodomain of HIV-1 gp41.

To execute the membrane fusion function, it is necessary for the fusion protein of the virus to penetrate into the hydrophobic milieu of membrane bilayer. Hence identification of the region(s) of the ectodomain of viral fusion proteins involved in the membrane insertion and their interaction with the rest of the fusion protein in the membrane would be important for the mechanistic study of membrane fusion. To this end, we examined membrane activity of the fusion peptide, and the ectodomain protein with or without the fusion peptide domain of HIV-1 gp41 by several biophysical measurements. The results revealed that the ectodomain protein containing the fusion peptide domain had higher membrane-perturbing activity and deeper membrane insertion, while the construct lacking the fusion peptide domain had much lower membrane activity. Strikingly, the N-terminal heptad repeat region was found to be induced deeper into the membrane by the fusion peptide, consistent with the role of the latter in the membrane penetration. We concluded that the fusion peptide is the only stretch of gp41 ectodomain that embeds deeply in the membrane interior in the prefusion stage. The function of fusion peptide in terms of membrane interaction and the implications of its interplay with other domains of gp41 on the membrane fusion cascade were discussed.

Construction and characterization of insect cell-derived influenza VLP: cell binding, fusion, and EGFP incorporation.

We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

Electrochemistry and Rapid Electrochromism Control of MoO3/V2O5 Hybrid Nanobilayers.

MoO3/V2O5 hybrid nanobilayers are successfully prepared by the sol-gel method with a spin- coating technique followed by heat -treatment at 350 °C in order to achieve a good crystallinity. The composition, morphology, and microstructure of the nanobilayers are characterized by a scanning electron microscope (SEM) and X-ray diffractometer (XRD) that revealed the a grain size of around 20-30 nm, and belonging to the monoclinic phase. The samples show good reversibility in the cyclic voltammetry studies and exhibit an excellent response to the visible transmittance. The electrochromic (EC) window displayed an optical transmittance changes (ΔT) of 22.65% and 31.4% at 550 and 700 nm, respectively, with the rapid response time of about 8.2 s for coloration and 6.3 s for bleaching. The advantages, such as large optical transmittance changes, rapid electrochromism control speed, and excellent cycle durability, demonstrated in the electrochromic cell proves the potential application of MoO3/V2O5 hybrid nanobilayers in electrochromic devices.

Development of Thermally Conductive Polyurethane Composite by Low Filler Loading of Spherical BN/PMMA Composite Powder.

The issue of electronic heat dissipation has received much attention in recent times and has become one of the key factors in electronic components such as circuit boards. Therefore, designing of materials with good thermal conductivity is vital. In this work, a thermally conductive SBP/PU composite was prepared wherein the spherical h-BN@PMMA (SBP) composite powders were dispersed in the polyurethane (PU) matrix. The thermal conductivity of SBP was found to be significantly higher than that of the pure h-BN/PU composite at the same h-BN filler loading. The SBP/PU composite can reach a high thermal conductivity of 7.3 Wm-1 K-1 which is twice as high as that of pure h-BN/PU composite without surface treatment in the same condition. This enhancement in the property can be attributed to the uniform dispersion of SBP in the PU polymer matrix that leads to a three-dimensional continuous heat conduction thereby improving the heat diffusion of the entire composite. Hence, we provide a valuable method for preparing a 3-dimensional heat flow path in polyurethane composite, leading to a high thermal conductivity with a small amount of filler.

Decreased Amount of Supporting Alveolar Bone at Single-Rooted Premolars Is Under Estimated by 2D Examinations.

The purpose of this study was to relate the proportions of bone-supported root length of a 2D view into the amount of a 3D bone-attached root surface area (BA-RSA) by using a dental laser scanner examination. White-light 3D scanning technology was used to probe 36 maxillary and 35 mandibular single-rooted premolars. The bone-supported height (BSH) and BA-RSA at designated levels (95-25%) were compared using statistical t tests. The 100% BSH and BA-RSA of the maxillary/mandibular premolars were 12.6 ± 1.60 mm/13.45 ± 1.47 mm (p < 0.05) and 220.78 ± 35.31 mm2/199.51 ± 26.33 mm2 (p < 0.01), respectively. Approximately 79-80%, 59-60%, and 35-36% premolars 2D BSH remained in comparison to 75%, 50%, and 25% 3D BA-RSA preservation, respectively. However, corresponding to a 75%, 50%, and 25% 2D BSH reserve, premolars retained 67-68%, 39-41%, and 15-17% 3D BA-RSA, respectively. When taking 1.0 mm connective tissue attachment into account, 60% 3D BA-RSA and 50% 2D BSH loss were noted at the 5.1-5.4 mm clinical attachment level. Assigning a periodontal prognosis and determining the severity of periodontitis for premolars with alveolar bone loss based on 3D's or 2D's measurement is inconsistent.

Ultrafast dynamics of quasiparticles and coherent acoustic phonons in slightly underdoped (BaK)Fe2As2.

We have utilized ultrafast optical spectroscopy to study carrier dynamics in slightly underdoped (BaK)Fe2As2 crystals without magnetic transition. The photoelastic signals due to coherent acoustic phonons have been quantitatively investigated. According to our temperature-dependent results, we found that the relaxation component of superconducting quasiparticles persisted from the superconducting state up to at least 70 K in the normal state. Our findings suggest that the pseudogaplike feature in the normal state is possibly the precursor of superconductivity. We also highlight that the pseudogap feature of K-doped BaFe2As2 is different from that of other iron-based superconductors, including Co-doped or P-doped BaFe2As2.

Heterojunction of Zinc Blende/Wurtzite in Zn1-xCdxS Solid Solution for Efficient Solar Hydrogen Generation: X-ray Absorption/Diffraction Approaches.

In the past decade, inorganic semiconductors have been successfully demonstrated as light absorbers in efficient solar water splitting to generate chemical fuels. Pseudobinary semiconductors Zn1-xCdxS (0≤x≤1) have exhibited a superior photocatalytic reactivity of H2 production from splitting of water by artificial solar irradiation without any metal catalysts. However, most studies had revealed that the extremely high efficiency with an optimal content of Zn1-xCdxS solid solution was determined as a result of elevating the conduction band minimum (CBM) and the width of bandgap. In addition to corresponding band structure and bandgap, the local crystal structure should be taken into account as well to determine its photocatalytic performance. Herein, we demonstrated the correlations between the photocatalytic activity and structural properties that were first studied through synchrotron X-ray diffraction and X-ray absorption spectroscopy. The crystal structure transformed from zinc blende to coexisted phases of major zinc blende and minor wurtzite phases at a critical point. The heterojunction formed by coexistence of zinc blende and wurtzite phases in the Zn1-xCdxS solid solution can significantly improve the separation and migration of photoinduced electron-hole pairs. Besides, X-ray absorption spectra and UV-vis spectra revealed that the bandgap of the Zn0.45Cd0.55S sample extended into the region of visible light because of the incorporation of Cd element in the sample. These results provided a significant progress toward the realization of the photoelectrochemical mechanism in heterojunction between zinc blende and wurtzite phases, which can effectively separate the charge-carriers and further suppress their recombination to enhance the photocatalytic reactivity.

Kinetics study on the HIV-1 ectodomain protein quaternary structure formation reveals coupling of chain folding and self-assembly in the refolding cascade.

Entry of HIV-1 into the target cell is mediated by the envelope glycoprotein consisting of noncovalently associated surface subunit gp120 and transmembrane subunit gp41. To form a functional gp41 complex, the protein undergoes hairpin formation and self-assembly. The fusion event can be inhibited by gp41-derived peptides at nanomolar concentration and is highly dependent on the time of addition, implying a role of folding kinetics on the inhibitory action. Oligomerization of the gp41 ectodomain was demonstrated by light scattering measurements. Kinetic study by stopped-flow fluorescence and absorption measurements (i) revealed a multistate folding pathway and stable intermediates; (ii) showed a dissection of fast and slow components for early and late stages of folding, respectively, with 3 orders of magnitude difference in the time scale; (iii) showed the slow process was attributed to misfolding and unzipping of the hairpin; and (iv) showed retardation of the native hairpin formation is assumed to lead to coupling of the correctly registered hairpin and self-assembly. This coupling allows the deduction on the time scale of intrachain folding (0.1-1 s) for the protein. The folding reaction was illustrated by a free energy profile to explain the temporal dichotomy of fast and slow steps of folding as well as effective inhibition by gp41-derived peptide.

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system.

We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.