Establishment of killer whale (Orcinus orca) primary fibroblast cell cultures and their transcriptomic responses to pollutant exposure.

Populations of killer whale (Orcinus orca) contain some of the most polluted animals on Earth. Yet, the knowledge on effects of chemical pollutants is limited in this species. Cell cultures and in vitro exposure experiments are pertinent tools to study effects of pollutants in free-ranging marine mammals. To investigate transcriptional responses to pollutants in killer whale cells, we collected skin biopsies of killer whales from the Northern Norwegian fjords and successfully established primary fibroblast cell cultures from the dermis of 4 out of 5 of them. Cells from the individual with the highest cell yield were exposed to three different concentrations of a mixture of persistent organic pollutants (POPs) that reflects the composition of the 10 most abundant POPs found in Norwegian killer whales (p,p'-DDE, trans-nonachlor, PCB52, 99, 101, 118, 138, 153, 180, 187). Transcriptional responses of 13 selected target genes were studied using digital droplet PCR, and whole transcriptome responses were investigated utilizing RNA sequencing. Among the target genes analysed, CYP1A1 was significantly downregulated in the cells exposed to medium (11.6 µM) and high (116 µM) concentrations of the pollutant mixture, while seven genes involved in endocrine functions showed a non-significant tendency to be upregulated at the highest exposure concentration. Bioinformatic analyses of RNA-seq data indicated that 13 and 43 genes were differentially expressed in the cells exposed to low and high concentrations of the mixture, respectively, in comparison to solvent control. Subsequent pathway and functional analyses of the differentially expressed genes indicated that the enriched pathways were mainly related to lipid metabolism, myogenesis and glucocorticoid receptor regulation. The current study results support previous correlative studies and provide cause-effect relationships, which is highly relevant for chemical and environmental management.

Seroprevalence of Various Viral Diseases in Tamil Nadu, India.

Introduction: Virus-borne diseases have recently gained significant public health importance. Viruses infect several hosts, including animal reservoirs, evolve quickly, and recombine emerging and reemerging to pose recurring dangers to humans. The Viral Research and Diagnostic Laboratory (VRDL) located at Government Theni Medical College, Theni, Tamil Nadu, conducts the diagnosis of common virus infections. Methods: From January 2018 to December 2022, the VRDL received whole blood sera samples from 84,059 patients suspected of having various viral illnesses. The enzyme-linked immunosorbent assay was used to detect viral infections in all of the samples. Results: A total of 84,059 individuals suspected for various viral infections have been tested and out of these 4948 (5.88%) cases have been reported to be positive and among them, the dengue virus is predominantly followed by, hepatitis B virus, chikungunya virus, hepatitis C virus, hepatitis A virus, hepatitis E virus, hepatitis B virus, herpes simplex virus, cytomegalovirus, and rubella virus. Conclusion: The issue of emerging and re-emerging infectious illnesses, particularly those caused by viruses, has grown in importance in public health. Timely action combined with proper information and the ability to diagnose infections may save many lives.

Facile manufacturing of fused-deposition modeled composite scaffolds for tissue engineering-an embedding model with plasticity for incorporation of additives.

The fused-deposition modeling (FDM) process is carried out at an elevated temperature, preventing the addition of biological factors, drugs, bioactive compounds, etc, during fabrication. To overcome this disadvantage, a 3D interlinked porous polylactic acid (PLA) scaffold was fabricated by FDM, followed by the embedding of a polycaprolactone (PCL) scaffold into the pores of the PLA at room temperature, yielding a PLA-PCL scaffold. In addition, PLA-PCL scaffolds with nanohydroxyapatite (PLA-PCL-nHAP) and multiwalled carbon nanotubes (PLA-PCL-MWCNT) were also fabricated. Here, the FDM-fabricated PLA scaffold functions as the structural component, whereas the embedded PCL scaffold acts as the functional component, which provides a the ability to functionalize the scaffolds with the desired chemical or biological materials. The embedding process is straightforward, cost effective, and does not require sophistication. A mechanical characterization of the scaffolds suggests that the Young's modulus of the PLA-PCL scaffold (16.02 MPa) was higher than that of the FDM-fabricated PLA (9.98 MPa) scaffold, by virtue of embedded PCL matrix. In addition, finite element analysis showed that the von Mises stress on a mandible with scaffolds was 4.04 MPa, whereas for a mandible with a defect, it was 6.7 MPa, confirming the stress distribution efficiency and mechanical stability of these scaffolds. Furthermore, field emission-scanning electron microscope analysis implied the presence of interlinked porous structures with pore diameters of 50 µm to 300 µm. X-ray diffraction results revealed an increased crystallinity (%) in the embedded models (PLA-PCL, PLA-PCL-nHAP and PLA-PCL-MWCNT), compared to a PLA printed scaffold. Additionally, Raman analysis revealed that the embedding process did not cause chemical alterations in the polymeric chains. In vitro analysis with human osteoblasts demonstrated the osteoconductive nature of the scaffold, which supported mineralization. In brief, the advantage of our model is that it helps to overcome the difficulties of manufacturing a filament with the desired additives for FDM, and offers the ability to incorporate the desired concentrations of heat-labile bioactive molecules during the embedding process at ambient temperatures.

Fabrication of tri-layered electrospun polycaprolactone mats with improved sustained drug release profile.

Modulation of initial burst and long term release from electrospun fibrous mats can be achieved by sandwiching the drug loaded mats between hydrophobic layers of fibrous polycaprolactone (PCL). Ibuprofen (IBU) loaded PCL fibrous mats (12% PCL-IBU) were sandwiched between fibrous polycaprolactone layers during the process of electrospinning, by varying the polymer concentrations (10% (w/v), 12% (w/v)) and volume of coat (1 ml, 2 ml) in flanking layers. Consequently, 12% PCL-IBU (without sandwich layer) showed burst release of 66.43% on day 1 and cumulative release (%) of 86.08% at the end of 62 days. Whereas, sandwich groups, especially 12% PCLSW-1 & 2 (sandwich layers-1 ml and 2 ml of 12% PCL) showed controlled initial burst and cumulative (%) release compared to 12% PCL-IBU. Moreover, crystallinity (%) and hydrophobicity of the sandwich models imparted control on ibuprofen release from fibrous mats. Further, assay for cytotoxicity and scanning electron microscopic images of cell seeded mats after 5 days showed the mats were not cytotoxic. Nuclear Magnetic Resonance spectroscopic analysis revealed weak interaction between ibuprofen and PCL in nanofibers which favors the release of ibuprofen. These data imply that concentration and volume of coat in flanking layer imparts tighter control on initial burst and long term release of ibuprofen.

Progestin-based contraception regimens modulate expression of putative HIV risk factors in the vaginal epithelium of pig-tailed Macaques.

PROBLEM: In women, the use of progestin-based contraception may increase the risk of vaginal HIV acquisition. We previously showed in macaques that there is a significantly higher simian-human immunodeficiency virus (SHIV) acquisition rate in the luteal phase of the menstrual cycle, which presents a naturally high-progesterone state, and this may be attributable to altered expression of innate immune factors. We hypothesized that progestin-based contraception, especially depot medroxyprogesterone acetate (DMPA), would, in a similar way, affect mucosal immune factors that influence HIV acquisition risk. METHOD OF STUDY: We used a pig-tailed macaque model to evaluate the effects of two progestin-based contraceptives, DMPA, and levonorgestrel (LNG)/ethinyl estradiol (EE)-based combined oral contraceptives (COCs), on innate mucosal factors. We compared the vaginal epithelial thickness data from previous studies and used cytokine profiling and microarray analysis to evaluate contraception-induced molecular changes in the vagina. RESULTS: The administration of DMPA caused a reduction in the thickness of the vaginal epithelium relative to that of the follicular or luteal phase. DMPA also induced a significant increase in vaginal levels of the anti-inflammatory cytokine IL-10. Both DMPA- and LNG-based contraception induced a signature of gene expression similar to that of the luteal phase, only more exacerbated, including widespread downregulation of antiviral genes. CONCLUSION: The use of progestin-based contraception might engender a milieu that poses an increased risk of HIV acquisition as compared to both the luteal and follicular phases of the menstrual cycle.

Abnormal amyloid ß42 expression and increased oxidative stress in plasma of CKD patients with cognitive dysfunction: A small scale case control study comparison with Alzheimer's disease.

BACKGROUND: Cognitive dysfunction has been increasingly recognized in chronic kidney disease (CKD) patients. Senile plaques are important pathophysiological characteristic of cognitive dysfunction. The major component of plaques is the amyloid ß (Aß) peptide released from proteolytic cleavage of amyloid precursor protein (APP). Plasma Aß has been a focus of the growing literature on blood based biomarkers for cognitive dysfunction. Oxidative stress is prevalent in CKD and it plays an important role in cognitive dysfunction. Increased oxidative stress leads to cause cleavage of APP and Aß production. The aim of this study is to assess the antioxidant status and Aß42 levels in plasma of CKD patients with cognitive dysfunction compared to CKD without cognitive dysfunction. METHODS: A total of 60 subjects divided into 30 CKD without cognitive dysfunction and 30 CKD with cognitive dysfunction based on neuropsychological assessment tests. To compare antioxidant status and Aß42 levels in plasma, the following groups such as healthy subjects (n = 30), normocytic normochromic anemia (n = 30) and Alzheimer's disease (AD, n = 10) patients were also maintained. Plasma Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), Reduced glutathione (GSH) and lipid peroxidation (LPO) were determined by spectrophotometrically. Aß level was determined by immunoblotting method. The parameters were statistically compared with healthy, normocytic normochromic anemia and AD subjects. RESULTS: Like AD subjects, significantly increased Aß and LPO level while decreased SOD, CAT, GPx and GSH levels were observed in plasma of CKD patients with cognitive dysfunction when compared to healthy, CKD without cognitive dysfunction and normocytic normochromic anemic subjects. CONCLUSION: Results suggest that elevated plasma oxidative stress and Aß were seen in CKD patients with cognitive dysfunction may be attributed to pathological changes within the brain.

Transcriptomic changes in relation to early-life events in the gilthead sea bream (Sparus aurata).

BACKGROUND: Teleosts are exposed to a broad range of external stimuli, which may be either of acute or chronic nature. The larval phase of certain fish species offer a unique opportunity to study the interactions between genes and environmental factors during early life. The present study investigates the effects of early-life events, applied at different time points of early ontogeny (first feeding, flexion and development of all fins; Phase 1) as well as on the subsequent juvenile stage after the application of an additional acute stressor (Phase 2) in the gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. Animal performance, the cortisol response and gene expression patterns during early development as well as on the subsequent phases (juveniles) after the application of additional acute stressors were investigated. RESULTS: Significant differences on fish performance were found only for juveniles exposed to early-life events at the phase of the formation of all fins. On the transcriptome level distinct expression patterns were obtained for larvae as well as for juveniles with the most divergent expression pattern found to be again at the phase of the development of all fins, which showed to have also an impact later on in the acute stress response of juveniles. CONCLUSIONS: The present study showed that applying an early-life protocol, characterized by the unpredictable, variable and moderate intensity of the applied stimuli provides a relative realistic model to evaluate the impact of daily aquaculture practices on fish performance. In addition, the power of investigating global gene expression patterns is shown, providing significant insights regarding the response of early-life events during development and as juveniles after the application of extra acute stressors.

Influence of Field Position on Rugby League Players Requiring Shoulder Reconstruction.

A consecutive series of patients sustaining their index anterior instability while playing Rugby League and requiring shoulder reconstruction was retrospectively reviewed and evaluated. The details of their on-field position and hand dominance at time of injury, the side of injury, and mechanism of injury were collated and statistically analyzed. A total of 173 Bankart repairs were performed on 132 patients, and 102 players had one injury while 30 had 2 or more injuries. Players are more likely to injure their non-dominant side (P=0.009) in the first-time injury. Whereas second and subsequent injuries were not associated with a particular side (P=0.81). Applying a Bonferroni correction to the chi-squared goodness-of-fit test of position at time of injury revealed players on the wing had a significantly lower frequency of injury (P<0.001), whereas the full back had a significantly higher frequency of injury (P<0.001). 30 players sustained 69 re-dislocations with the lock and fullback positions incurring significantly more re-injuries and the wing position fewer re-injuries than expected (P<0.006). Understanding which player positions on the rugby league field are more likely to sustain an anterior instability will assist medical and support staff to optimize the pre-season conditioning and post surgical rehabilitation of players.

Macaque models of enhanced susceptibility to HIV.

There are few nonhuman primate models of enhanced HIV susceptibility. Such models can improve comprehension of HIV acquisition risk factors and provide rigorous testing platforms for preclinical prevention strategies. This paper reviews past, current, and proposed research on macaque HIV acquisition risk models and identifies areas where modeling is significantly lacking. We compare different experimental approaches and provide practical considerations for designing macaque susceptibility studies. Modifiable (mucosal and systemic coinfections, hormonal contraception, and rectal lubricants) and non-modifiable (hormonal fluctuations) risk factors are highlighted. Risk acquisition models via vaginal, rectal, and penile challenge routes are discussed. There is no consensus on the best statistical model for evaluating increased susceptibility, and additional research is required. The use of enhanced susceptibility macaque models would benefit multiple facets of the HIV research field, including basic acquisition and pathogenesis studies as well as the vaccine and other biomedical preventions pipeline.

Rectal application of a highly osmolar personal lubricant in a macaque model induces acute cytotoxicity but does not increase risk of SHIV infection.

BACKGROUND: Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication in vitro. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although in vivo evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15. METHODS: Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIVSF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure. RESULTS: Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID50 (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models). CONCLUSIONS: Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the in vivo evaluation of lubricants with regards to HIV transmission.

Short communication: Viremic control is independent of repeated low-dose SHIVSF162p3 exposures.

The repeat low-dose virus challenge model is commonly used in nonhuman primate studies of HIV transmission and biomedical preventions. For some viruses or challenge routes, it is uncertain whether the repeated exposure design might induce virus-directed innate or adaptive immunity that could affect infection or viremic outcomes. Retrospective cohorts of male Indian rhesus (n=40) and female pigtail (n=46) macaques enrolled in repeat low-dose rectal or vaginal SHIV(SF162p3) challenge studies, respectively, were studied to compare the relationship between the number of previous exposures and peak plasma SHIV RNA levels or viral load area under the curve (AUC), surrogate markers of viral control. Repeated mucosal exposures of 10 or 50 TCID50 of virus for rectal and vaginal exposures, respectively, were performed. Virus levels were measured by quantitative reverse-transcriptase real-time PCR. The cumulative number of SHIV(SF162p3) exposures did not correlate with observed peak virus levels or with AUC in rectally challenged rhesus macaques [peak: rho (ρ)=0.04, p=0.8; AUC: ρ=0.33, p=0.06] or vaginally challenged pigtail macaques (peak: ρ=-0.09, p=0.7; AUC: ρ=0.11, p=0.6). Infections in these models occur independently of exposure history and provide assurance that neither inoculation route nor number of exposures required for infection correlates with postinfection viremia. These data also indicate that both the vaginal and rectal repeated low-dose virus exposure models using SHIV(SF162p3) provide a reliable system for nonhuman primate studies.

SHIV susceptibility changes during the menstrual cycle of pigtail macaques.

BACKGROUND: Hormonal changes during menstrual cycling may affect susceptibility to HIV. METHODS: We determined the simian human immunodeficiency virus (SHIV) acquisition time point in 43 cycling pigtail macaques infected by repeated vaginal virus exposures initiated randomly in the cycle. RESULTS: SHIV infection was first detected in the follicular phase in 38 macaques (88%), and in the luteal phase in five macaques (12%), indicating a statistically significant timing difference. Assuming a 7-day eclipse phase, most infections occurred during or following a high-progesterone period associated with menstruation, vaginal epithelium thinning, and suppressed mucosal immunity. CONCLUSIONS: This raises questions whether other high-progesterone conditions (pregnancy, hormonal contraception) similarly affect HIV risk.

Non-human primate models of hormonal contraception and HIV.

PROBLEM: Recent concerns that hormonal contraception (HC) may increase risk of HIV acquisition has led to keen interest in using non-human primates (NHP) to understand the underlying mechanism and the magnitude of the risk. This is, in part, because some experiments which would be difficult or logistically impossible in women are more easily conducted in NHP. METHOD OF STUDY: NHP models of HIV can inform HIV acquisition and pathogenesis research and identify and evaluate biomedical preventions and treatments for HIV/AIDS. Widely used species include rhesus, pigtail, and cynomolgous macaques. RESULTS: This paper reviews past, current and proposed NHP research around the intersection of HIV and HC. CONCLUSION: NHP research may lead to the identification of hormonally regulated biomarkers that correlate with HIV-acquisition risk, to a ranking of existing or next-generation HC along an HIV-acquisition risk profile, and inform research around new biomedical preventions for HIV.

Evaluation of pigtail macaques as a model for the effects of copper intrauterine devices on HIV infection.

BACKGROUND: Long-acting, hormonal contraception may increase HIV risk. Copper intrauterine devices (IUDs) could serve as non-hormonal alternatives. We pilot a pigtail macaque model for evaluating HIV susceptibility factors during copper IUD use. METHODS: Frameless and flexible GyneFix(®) copper IUDs were surgically implanted into three SHIVSF 162p3 -positive macaques via hysterotomy and monitored for up to 4 months. Four macaques served as non-IUD controls. RESULTS: All animals retained the devices without complications. No consistent change in vaginal viral RNA or inflammatory cytokines was seen. Two animals had altered menstrual cycles and experienced marked thinning of vaginal epithelium after IUD insertion. Histological examination of uterine tissue at necropsy revealed endometrial ulceration and lymphocytic inflammation with glandular loss at sites of direct IUD contact. CONCLUSIONS: Although the need for insertion surgery could limit its usefulness, this model will allow studies on copper IUDs and SHIV shedding, disease progression, and HIV susceptibility factors.

Role of transmitted Gag CTL polymorphisms in defining replicative capacity and early HIV-1 pathogenesis.

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.

Effect of cross-fostering on renal anti-oxidant/oxidant status and development of hypertension in spontaneously hypertensive rats.

1. The hypotensive effect of cross-fostering in spontaneously hypertensive rats (SHR) is thought to involve adjustments in renal function. However, its association with renal anti-oxidant/oxidant balance during cross-fostering is not known. 2. The present study examined the effect of cross-fostering and in-fostering of 1-day-old offspring between SHR and Wistar-Kyoto (WKY) dams on renal anti-oxidant/oxidant status and systolic blood pressure (SBP). Renal anti-oxidant/oxidant status and SBP were determined in the offspring from 4-16 weeks of age. 3. Cross-fostered SHR had significantly lower SBP than in-fostered SHR at 6, 8 and 12 weeks, but not at 16 weeks (127 ± 1 vs 144 ± 2, 138 ± 1 vs 160 ± 1, 174 ± 2 vs 184 ± 2 and 199 ± 2 vs 194 ± 3 mmHg at 6, 8, 12 and 16 weeks, respectively). No differences in SBP were evident between cross-fostered and in-fostered WKY rats. There were no significant differences in levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyl and total anti-oxidant status (TAS) or superoxide dismutase, catalase, glutathione peroxidase (GPx), glutathione S-transferase and glutathione reductase activity between cross-fostered and in-fostered SHR or WKY offspring. However, compared with WKY rats, catalase activity was higher at 6 and 16 weeks, TAS was higher at 16 weeks and GPx activity and TBARS were lower at 16 weeks in SHR. 4. It appears that cross-fostering of SHR offspring to WKY dams during the early postnatal period causes a transient delay in the rise in blood pressure in SHR and that this does not involve the renal anti-oxidant/oxidant system.

High susceptibility to repeated, low-dose, vaginal SHIV exposure late in the luteal phase of the menstrual cycle of pigtail macaques.

Fluctuations in susceptibility to HIV or SHIV during the menstrual cycle are currently not fully documented. To address this, the time point of infection was determined in 19 adult female pigtail macaques vaginally challenged during their undisturbed menstrual cycles with repeated, low-dose SHIV(SF162P3) exposures. Eighteen macaques (95%) first displayed viremia in the follicular phase, as compared with 1 macaque (5%) in the luteal phase (P < 0.0001). Due to a viral eclipse phase, we estimated a window of most frequent virus transmission between days 24 and 31 of the menstrual cycle, in the late luteal phase. Thus, susceptibility to vaginal SHIV infection is significantly elevated in the second half of the menstrual cycle when progesterone levels are high and when local immunity may be low. Such susceptibility windows have been postulated before but not definitively documented. Our data support the findings of higher susceptibility to HIV in women during progesterone-dominated periods including pregnancy and contraceptive use.

Large changes in the CRAC segment of gp41 of HIV do not destroy fusion activity if the segment interacts with cholesterol.

The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol.