Carrageenan Gel Research Articles

Efficacy Study of Carrageenan as an Alternative Infused Material (Filler) in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Porous 3D Scaffold

Polymeric porous 3D scaffold plays an important role in culturing mammalian cells asex vivomodel. However, the scaffold used is ineffective due to its structural and cell acceptability weaknesses. Therefore, this research attempts to overcome the weaknesses by using carrageenan from red seaweedKappaphycus alvareziias an alternative infused material (filler) of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) porous 3D scaffold. The 3D scaffold was conventionally fabricated using the solvent-casting particulate-leaching (SCPL) method. Carrageenan was later infused into 3D porous scaffolds under vacuum pressure and freeze-drying process. Five carrageenan concentrations were prepared and its physicochemical properties such as pH and viscosity were carried out on each concentration to determine the best solutions to produce a new composite 3D structure. The preliminary result shows that carrageenan concentrations of 2, 4, and 6% (w/v) were considered the best solutions for the infusion process due to its stable rheology properties. The pH and viscosity profiles of three selected carrageenan solutions were exhibited in the range of 9.00–9.20 and 0.047–1.144 Pa·s, respectively. Moreover, the incorporated carrageenan gel fraction was in the range of 4.30% to 14.95% (w/w) which was determined by gravimetric analysis and dye staining method (visual assessment). The well-infused carrageenan 3D scaffold was further characterized based on its internal morphology and degradability study. The vertical cross-sections of the scaffolds revealed homogeneous accumulation of dried gelatinous carrageenan which was covered throughout its pores wall. The degradation rate (K) of the carrageenan infused 3D scaffold was between0.01±1.66(mg/day) and0.03±3.23(mg/day). The higher the carrageenan concentration used, the faster the degradation rate occurring (p<0.05). The 3D infused scaffold of 4% (w/v) carrageenan concentration (S2) produced a moderate degradation rate of0.02±1.55(mg/day) with a sustained structural integrity up to 28 days. The carrageenan infused scaffold of 4% (w/v) was demonstrated to be the best 3D structure for a long-term cell culture (>2 weeks). In conclusion, the usage of carrageenan as a composite material exhibits its great potential to be used in tissue engineering application and 3D cell culture model.

Production of κ‐carrageenan beads by prilling process

AbstractThe objective of this study is to prepare, evaluate, and understand carrageenan gelation. By using these properties of κ‐carrageenan, beads are created by thermal gelation. A warm κ‐carrageenan solution is extruded dropwise into a cold air column; the particles are collected. The relationship is studied between the process variables on the shape and size of κ‐carrageenan drops after gelation is established with the aid of image analysis. Thanks to these measurements, a model is developed to present satisfactory performance in industrial application.Mathematical models for κ‐carrageenan prilling, its simulated results, the parameters of equipment for the innovated process, and its application are addressed.

Covalent immobilization of cellulase in application of biotransformation of ginsenoside Rb1

Cellulase was immobilized on the surface of carrageenan beads activated by using polyethyleneimine and glutaraldehyde, then immobilized cellulase was used to transform ginsenoside Rb1. In this work, we studied the functionalization of carrageenan with amine terminal functional group for the covalent immobilization of cellulase. The process of immobilization was performed by amination process on the surface of the carrageenan gel beads with polyethylenimine followed by activation with glutaraldehyde to finally immobilize the enzyme. Different factors affecting the amination and activation processes were studied and their effects on the catalytic activity of the immobilized cellulase were followed, then Fourier transform infrared spectroscopy and scanning electron microscopy were applied to verify the process. Furthermore, the optimum conditions of transformation of ginsenoside Rb1 were also studied and showed better stability for the immobilized enzyme. The best results were obtained using carrageenan as support and covalent binding method, which consisted of amination process (0.5% PEI (pH 8))) for 3h and 3% GA in activation process for 3h. These experiments gave optimum reaction condition of temperature at 60°C and pH 5.0. Enzyme after immobilization exhibited high activity with broader pH and temperature range. Full conversion and kinetic parameters Km and Vmax for them were also studied. The Km of immobilized enzyme, 3.64mM, is lower than free one, 4.82mM; and the Vmax of immobilized enzyme, 0.04mM, is lower than free one, 0.06mM. Results detected also show after using for 5 consecutive times, the immobilized enzyme can retain 60% of its initial activity.

Drying characteristics and some properties of spouted bed dried semi-refined carrageenan

The study was focused on the evaluation of spouted bed drying as a method for achieving dried carrageenan gels suitable for grinding, in order to obtain a final product in the form of a powder. Cubes formed from semi-refined carrageenan gels K12, K73, and their blends with carrageenan 92S (iota) were dried in a spouted bed at three temperatures: 60, 100, and 150 °C. Functional properties of the dried product were assessed in terms of density (bulk, apparent, and true), water activity, color parameters, rheological properties, and solubility. The results revealed that the decrease in the moisture ratio with time during spouted bed drying of carrageenan gels can be described by the Page model, which was characterized by satisfactorily fitting parameters such as R2, RMSE, and Ve. On the other hand, the decrease in the spouted bed volume ratio versus moisture content had linear characteristics, and depended significantly only on the kind of material. The increase in hot air temperature from 60 to 150 °C decreased the drying time for all types of carrageenan gels by ca 50%, contributing to lower equilibrium moisture content and water activity, as well as a lighter color of the dried product, apart from K73 + iota. However, drying at 100 °C provided product with the best solubility and high gelling ability. The differences in the final values of the bulk and apparent densities of dried carrageenan samples were explained, taking into account the effect of temperature and the materials’ properties on the behavior of shrinkage.

Examining the effect of freezing on starch gelatinization during heating at high rates using online in situ hot-stage video-microscopy and differential scanning calorimetry

Despite its importance, little attention has been paid to the effect of freezing on starch gelatinization during subsequent heating. In an effort to address this problem, the effects of freezing and water accessibility in starch gelatinization during heating were analyzed in situ using hot-stage polarized-light video-microscopy. Native starch granules immersed in water, carrageenan gel or sucrose solution were heated at 15°C/min or frozen for 48h and heated at 15°C/min. Energy changes were measured using differential scanning calorimetry. Surface morphology was examined using scanning electron microscopy and atomic force microscopy. The results showed that freezing delayed the gelatinization degree and that the addition of carrageenan delayed the gelatinization process. That process was further delayed when sucrose was added. The enthalpy of gelatinization decreased accordingly (ΔHstarch−water−sucrose>ΔHstarch−water−carrageenan>ΔHstarch−water). The temperature range over which the degree of gelatinization differed between frozen and unfrozen samples was diminished when accessibility to water was reduced.

Gelling characteristics and rheology of kappa/iota-hybrid carrageenans extracted from Mastocarpus stellatus dried at different temperatures

The effect of the seaweed drying temperatures (35, 45, 55 and 85 °C), prior to the biopolymer extraction, on the rheological properties of kappa/iota-hybrid carrageenan (KI) gels in NaCl extracted from Mastocarpus stellatus seaweed is reported, together with the thermal stability of obtained gels. The average molecular mass (ranged between 2.25 × 106 and 1.02 × 106 kg kmol−1) of extracted KI, determined by gel permeation chromatography, and estimated intrinsic viscosities (ranged between 0.935 and 0.258 m3 kg−1), conducted in a capillary viscometer, decreased with increase of the seaweed drying temperatures. The parameters (K and a) of the Mark-Houwink-Sakurada (MHS) equation for KI are also reported. Thermo-rheological oscillatory measurements were conducted in a stress-controlled rheometer in order to clarify the kinetics of gel formation and to characterize the structure of the matured gels. Rheological results indicated a weakening of the KI gelling properties at smaller average molecular masses (i.e. larger seaweed drying temperatures, above 45 °C). Even so, KI samples exhibited stable and weak gel properties (i.e. elastic modulus <200 Pa). KI gels reached stability after 20 min of maturation. Cooling and heating profiles showed a strong temperature dependency. Gel setting temperatures significantly depended on the biopolymer content, whereas gel melting temperatures (68.0 ± 0.3 °C) were unvaried. No complete thermal stability was observed for gels formed with tested biopolymers, the most thermal stable being those prepared with KI with larger molecular masses (i.e. lower seaweed drying temperatures, below 45 °C).

Effect of starch source on gel properties of kappa-carrageenan-starch dispersions

Eight different starches (2%, w/w) were incorporated into dispersions containing various amount of kappa-carrageenan (0.2%, 0.6% and 1.0%, w/w), and the properties including morphology, rheology, texture and syneresis of gel from carrageenan and carrageenan-starch dispersions were examined and compared for elucidating the effect of starch source on gel properties of the dispersions. Results showed that the swelling of starch granules in carrageenan dispersions decreased with increasing concentration of carrageenan. Carrageenan dispersions incorporated with starch possessed high granule rigidity or low swelling power had high values of storage modulus and gel firmness. The presence of starch reduced the syneresis of carrageenan gels, and this was more profound for dispersions containing starch with high amylose content. At each concentration of carrageenan, swelling power of starch showed significant correlations with the storage modulus (r2 ≥ 0.590, p < 0.05) and firmness (r2 ≥ 0.890, p < 0.05) of carrageenan-starch dispersions. Results indicate that the presence of starch in carrageenan dispersion improves the gel strength and reduces the syneresis of carrageenan. Moreover, the amylose content of starch and the rigidity of gelatinized starch granules play important roles on gel properties of carrageenan-starch dispersions.

The effect of Ascophyllum nodosum (Ochrophyta) extract powder on the epibiosis of Kappaphycus alvarezii (Rhodophyta) commercially cultivated on floating rafts

Acadian marine plant extract powder (AMPEP), a product derived from Ascophyllum nodosum, has been widely used as a plant growth stimulant and recently has been tested on seaweeds with successful results on increasing growth and preventing epibiosis in Kappaphycus alvarezii cultivation. Although, the AMPEP ability to deal with epibiosis on seedlings cultivated in tubular nets and its commercial implications are still unknown. Between July 2012 and September 2013, three sampling periods were randomly selected at a Brazilian commercial farm in order to evaluate the effect of AMPEP on K. alvarezii daily growth rate, carrageenan yield and quality, and epibiont settling. Ten tubular nets containing three 100 g seedlings each (five treated with AMPEP and five not) were tied on floating rafts. Control samples displayed higher biomass of epibionts [0.76 ± 0.52 g (dry mass) tubular net−1] when compared to the AMPEP-treated samples [0.26 ± 0.27 g (dry mass) tubular net−1]. Carrageenan yield (44.65 ± 7.09 %) showed no significant difference between treatments (t = 0.46; p = 0.64), with larger percentages by the end of the third sampling period (F = 130.96, p < 0.001). When comparing AMPEP treatment with the control, carrageenan gel strength (252.14 ± 82.4 g cm−3) and viscosity (106.72 ± 45.11 cP) showed opposite results. The gel strength of AMPEP-treated samples was higher than their controls (t = 2.88; p = 0.007), while the gel viscosity of samples treated with AMPEP was lower than samples from their control (t = −2.4; p = 0.02). K. alvarezii daily growth rates between treatments were not different (t = 0.92; p = 0.36), exhibiting low commercial values probably due to herbivory. It was concluded that AMPEP was efficient against epibiont settlement on K. alvarezii cultivated on tubular nets tied on floating rafts maintaining favorable commercial results for carrageenan yield.

Estimation of thermal distribution in tissue-mimicking phantom made of carrageenan gel

To probe the temperature elevation effect caused by ultrasound, the use of a tissue-mimicking phantom was newly suggested. Carrageenan gel was adopted to realize not only the required transparency for visualization but also the acoustic characteristics similar to those of human tissue. To visualize the temperature elevation effect inside the phantom, thermochromic film with a critical temperature of discoloration was introduced. From the visualized image, the temperature elevation due to planar and focused ultrasound in the phantom was obtained quantitatively. To verify the suggested method, the bioheat equation was solved numerically by the Fourier transform method. The theoretical results show good agreement with experimental ones regarding the temperature distribution while plane and focused ultrasound was irradiated into the phantom.

Spatial and temporal variation in carrageenan yield and gel strength of cultivated Kappaphycus alvarezii (Doty) Doty in relation to environmental parameters in Palk Bay waters, Tamil Nadu, Southeast coast of India

Cultivated Kappaphycus alvarezii Doty (Doty) was analyzed for its dryweed quality (moisture and clean anhydrous weed (CAW) contents), carrageenan (semi-refined carrageenan (SRC)) yield, and gel strength with respect to environmental parameters (seawater temperature, salinity, nitrate, and phosphate) for 1 year from April 2012 to March 2013 from three different localities, viz., Mangadu, Munaikadu, and Vedalai, located in Palk Bay, Ramanathapuram District, Tamil Nadu, Southeast coast of India. Dryweed quality, viz., moisture content (35.00 ± 0.00 to 38.39 ± 0.19 %), clean anhydrous weed (39.26 ± 1.37 to 45.22 ± 0.57 %), and carrageenan (SRC) yield (33.30 ± 1.23 to 38.50 ± 0.20 to 0.30 %) with gel strength (379.05 ± 6.07 to 486.44 ± 5.57 g cm−2), was found to vary differently at all the sites during the study period. Two-way ANOVA showed that seawater temperature, salinity, and nitrate were significant spatially, while carrageenan (SRC) yield and gel strength were found to be significant (p = 0.05 level) both spatially and temporally. The carrageenan (SRC) obtained in this study met international specifications with respect to dryweed quality, carrageenan yield, and gel strength, suggesting its production indigenously for internal consumption as well as for export using the cultivated raw material and thus providing employment to the coastal rural people in India and earning foreign exchange.

Screening of mucoadhesive vaginal gel formulations

Rational design of vaginal drug delivery formulations requires special attention to vehicle properties that optimize vaginal coating and retention. The aim of the present work was to perform a screening of mucoadhesive vaginal gels formulated with carbomer or carrageenan in binary combination with a second polymer (carbomer, guar or xanthan gum). The gels were characterised using in vitroadhesion, spreadability and leakage potential studies, as well as rheological measurements (stress and frequency sweep tests) and the effect of dilution with simulated vaginal fluid (SVF) on spreadability. Results were analysed using analysis of variance and multiple factor analysis. The combination of polymers enhanced adhesion of both primary gelling agents, carbomer and carrageenan. From the rheological point of view all formulations presented a similar behaviour, prevalently elastic and characterised by loss tangent values well below 1. No correlation between rheological and adhesion behaviour was found. Carbomer and carrageenan gels containing the highest percentage of xanthan gum displayed good in vitro mucoadhesion and spreadability, minimal leakage potential and high resistance to dilution. The positive results obtained with carrageenan-xanthan gum-based gels can encourage the use of natural biocompatible adjuvants in the composition of vaginal products, a formulation field that is currently under the synthetic domain.

Antiviral Activity of κ-carrageenan Gel on Herpes Simplex Type І, a Human Study: An Open-Label Clinical Trial

Aim of study This study aimed to study the possible application of carrageenan on the illnesses related to the oral cavity by studying its effects in Viral infection presented with herpes simplex in human.(an-open label study) Subjects,materials and methods In this study patients presented with oral herpes simplex lesions were treated with carrageenan gel (0.9%)that was prepared .The gel was applied twice daily and the end-points of the study are the complete healing in absence of any discomfort within a short period of treatment. Results The results showed that two applications of κ-carrageenan per day accelerated the healing process within a short period (1-2 days) without complications or disfigurement in all patients. Conclusion κ –carrageenan besides ι-ond λ- carrageenan is useful in management of viral infection. κ –carrageenan is effective against herpes simplex labialis .

Enhancement of carrageenan gel quality in the commercially important tropical seaweed Eucheuma denticulatum (Rhodophyta), with postharvest treatment in low-nutrient conditions

Abstract The potential of postharvest culture in low-nutrient conditions was evaluated in terms of enhancement of carrageenan gel quality in the tropical seaweed Eucheuma denticulatum (“spinosum” of commerce) and as an alternative to industrial alkali treatment. Postharvest batch culture of seaweeds with either initially low or high nutrient concentrations (both ending with low nutrient concentrations) produced native carrageenans that had significantly higher gel strengths (48 g cm-2 in both treatments) than those treated with continuous culture in high nutrient concentrations (18 g cm-2). The gel strength of native carrageenans from batch-cultured seaweeds was also significantly higher than that of both native and alkali-treated carrageenans from seaweeds that had not undergone postharvest culture (11 and 29 g cm-2, respectively). The mechanism indicated for the enhancement of carrageenan gel quality involves stopping the production of precursor (galactose-6-sulfate) and converting the preexisting precursor to 3,6-anhydrogalactose units through in vivo enzymatic action. Postharvest batch culture with low nutrient concentrations is considered to be an eco-friendly alternative to industrial alkali treatment in the enhancement of carrageenan gel quality.

Raman Signal Enhancement Dependence on the Gel Strength of Ag/Hydrogels Used as SERS Substrates

A series of hydrogel samples composed of Ag nanoparticles dispersed in carrageenan gels have been prepared and used in SERS studies. These studies demonstrate the dependence of the enhancement of the SERS signal on the strength of the Ag/polysaccharide hydrogel. 2,2′-Dithiodipyridine was used as the analyte probe. Several strategies were employed in order to vary the gel strength. These include the increase of the polysaccharide content in the gel, the addition of KCl as cross-linker, and the variation of the type of carrageenan (κ, ι, λ) network. An increase in the gel strength originates an increase in the SERS enhancement observed. The results have been interpreted considering hot spots increase due to the formation of Ag particles nanojunctions as the biopolymer matrix tends to rearrange into stronger gels. This is the first report showing that there is a direct correlation between the gel strength of a hydrogel composite used as substrate and its analytical SERS sensitivity.

Rheology and structure of mixed kappa-carrageenan/iota-carrageenan gels

The rheology of mixed κ-carrageenan/ι-carrageenan gels was investigated in the presence of 75 or 150 mM K+. The addition of ι-carrageenan to κ-carrageenan led to an initial weak increase of the Young's modulus. A maximum occurred at concentrations ratios close to 1:1, followed by a weak decrease at higher ι-carrageenan concentrations. When κ- and ι-carrageenan were mixed at a constant total polysaccharide concentration, a plateau of the Young's modulus and a clear local minimum in the fracture strain in both compression and extension were identified at ratios between 2:1 and 4:3 (ι:κ). At all other ratios, the expected strong increase of the Young's modulus and decrease of the fracture strain were observed with an increasing κ-carrageenan fraction. A weak maximum of the fracture stress in compression and extension was observed at ratios between 1:1 and 1:2 (ι:κ). The structural and rheological findings support segregative phase-separation of mixed carrageenan gels in the presence of K+. Phase separation could be promoted through curing of the gel network of ι-carrageenan at temperatures above the coil-to-helix transition of κ-carrageenan.

Tissue Mimicking Phantom for Visualization of Temperature Elevation Caused by Ultrasound

초음파에 의한 온도상승효과를 조사하기 위한 생체 모의매질을 제안하였다. 생체와 유사한 음향특성을 가지며 내부온도분포의 관찰이 가능한 투명도를 확보하기 위하여 카라기난(carrageenan) 젤을 선택하였다. 매질 내부의 온도상승효과를 가시화 하기 위하여 온도에 따라 변색되는 시온필름을 사용하였으며 카라기난의 농도 및 첨가제로 사용한 수크로스(sucrose)의 농도에 따른 생체 모의매질의 음향특성을 조사하였다. 그 결과, 카라기난 및 수크로스의 농도를 조절함으로써 음향감쇠계수를 0.44~0.49 dB/cm/MHz의 범위에서 제어할 수 있었으며 음향임피던스의 경우 1.52~1.77 Mrayls의 범위에서 제어가능함을 확인했다. 제안된 생체 모의매질은 첨가제 및 카라기난의 농도에 따라 생체조직에 대응되는 음향특성을 갖도록 제어할 수 있었으며 초음파에 의한 매질내부의 온도상승 관찰이 가능하였다. 제안된 모의매질을 이용하여 평면 및 집속 초음파에 의한 온도상승효과를 비침습적으로 가시화하여 확인함으로써 본 제안법의 유효성을 검증하였다. To probe the temperature elevation effect caused by ultrasound, a tissue mimicking phantom was newly suggested. A carrageenan gel was adopted to realize not only the required transparency for visualization but also acoustic characteristics similar to human tissue. To visualize the temperature elevation inside phantom, thermochromic film with a critical temperature of discoloration was introduced. Acoustic characteristics of the tissue mimicking phantom were examined when the concentrations of carrageenan and sucrose changed. As the results, the attenuation coefficient of the phantom could be controlled in the range of 0.44~0.49 dB/cm/MHz, and the acoustic impedance in the range of 1.52~1.77 Mrayls. We could control the acoustic characteristics of the phantom by different concentration of carrageenan and sucrose, and it was possible to examine the temperature elevation caused by ultrasound in the phantom. The suggested method was verified by noninvasively visualizing the temperature elevation due to planar and focused ultrasound using the fabricated phantom.

Effects of High Pressure and Addition of Sucrose on the Quality Improvement of Frozen-thawed Carrageenan Gels.

Effects of High Pressure and Addition of Sucrose on the Quality Improvement of Frozen-thawed Carrageenan Gels.

Effects of High Pressure and Addition of Sucrose on the Quality Improvement of Frozen-thawed Carrageenan Gels.

Effects of High Pressure and Addition of Sucrose on the Quality Improvement of Frozen-thawed Carrageenan Gels.

Effects of a commercial extract of the brown alga Ascophyllum nodosum on the biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and its carrageenan yield and gel quality cultivated in Brazil

Despite of its success, the carrageenan industry has had to cope with difficulties due to epiphyte infestations and diseases known as ice-ice. Some promising results in respect of biomass production, carrageenan yield, and protection epiphytes were obtained using a powdered extract of the brown alga Ascophyllum nodosum in Kappaphycus alvarezii seedling production. This study focused on the effects of the A. nodosum extract on the treatment of K. alvarezii cultivated on commercial floating rafts not only to evaluate improvements demonstrated in vitro but also the effects on the quality of the carrageenan. The seedlings were treated in an A. nodosum extract solution and placed alongside their controls on commercial floating rafts using the tubular net technique. Daily growth rate, carrageenan yield, gel strength, and gel viscosity were obtained over 20 and/or 40 days. After 20 days, daily growth rates showed no significant difference (p = 0.44), while the carrageenan yield was higher in samples that were treated with the A. nodosum solution (p < 0.001). After 40 days, both daily growth rate (p = 000.7) and carrageenan yield (p = 0.009) were higher in treated samples; however, gel strength was higher in control samples (p = 0.03) as viscosity was also highest in the samples which had not been treated (p < 0.001). The use of the A. nodosum extract treatment on the cultivation in situ of K. alvarezii was positive since after 40 days when the daily growth rate and carrageenan yield increased. In spite of the negative effect on the quality of the semi-refined carrageenan, the values were within commercial standards.

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.