Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.

Pushing myelination - developmental regulation of myosin expression drives oligodendrocyte morphological differentiation.

Oligodendrocytes are the central nervous system myelin-forming cells providing axonal electrical insulation and higher-order neuronal circuitry. The mechanical forces driving the differentiation of oligodendrocyte precursor cells into myelinating oligodendrocytes are largely unknown, but likely require the spatiotemporal regulation of the architecture and dynamics of the actin and actomyosin cytoskeletons. In this study, we analyzed the expression pattern of myosin motors during oligodendrocyte development. We report that oligodendrocyte differentiation is regulated by the synchronized expression and non-uniform distribution of several members of the myosin network, particularly non-muscle myosins 2B and 2C, which potentially operate as nanomechanical modulators of cell tension and myelin membrane expansion at different cell stages.This article has an associated First Person interview with the first author of the paper.

Acute and chronic demyelinated CNS lesions exhibit opposite elastic properties.

Increased deposition of extracellular matrix (ECM) is a known inhibitor of axonal regrowth and remyelination. Recent in vitro studies have demonstrated that oligodendrocyte differentiation is impacted by the physical properties of the ECM. However, characterization of the mechanical properties of the healthy and injured CNS myelin is challenging, and has largely relied on non-invasive, low-resolution methods. To address this, we have employed atomic force microscopy to perform micro-indentation measurements of demyelinated tissue at cellular scale. Analysis of mouse and human demyelinated brains indicate that acute demyelination results in decreased tissue stiffness that recovers with remyelination; while chronic demyelination is characterized by increased tissue stiffness, which correlates with augmented ECM deposition. Thus, changes in the mechanical properties of the acutely (softer) or chronically (stiffer) demyelinated brain might contribute to differences in their regenerative capacity. Our findings are relevant to the optimization of cell-based therapies aimed at promoting CNS regeneration and remyelination.

Preparation of Matrices of Variable Stiffness for the Study of Mechanotransduction in Schwann Cell Development.

Extracellular matrix (ECM) elasticity may direct cellular differentiation and can be modeled in vitro using synthetic ECM-like substrates with defined elastic properties. However, the effectiveness of such approaches depends on the selection of a range of elasticity and ECM ligands that accurately model the relevant tissue. Here, we present a cell culture system than can be used to study Schwann cell differentiation on substrates which model the changes in mechanical ECM properties that occur during sciatic nerve development.

Myelinating glia differentiation is regulated by extracellular matrix elasticity.

The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds.

High frequency of HIV-1 dual infections among HIV-positive individuals in Cameroon, West Central Africa.

OBJECTIVES: To determine the frequency of dual inter- and intra-subtype HIV-1 infection among a cohort of 64 longitudinally-studied, HIV-1-positive individuals in Yaoundé, Cameroon. METHODS: Blood was collected every 3-6 months for up to 36 months and RNA was extracted from plasma. Gag fragment (HxB2 location 1577-2040) was amplified by nested RT-PCR, and mixed-time-point Heteroduplex Assays (HDAs) were performed. As heteroduplexes in this assay indicate >or=5% genetic discordance in the gag fragment, their presence reveals dual infection. Results were confirmed by phylogenetic analysis. RESULTS: Heteroduplexes were generated by specimens of 10 subjects (15.6%). Kaplan-Meier nonparametric estimate of maintenance of single infection was calculated; the rate/year of a 2 infection was found to be approximately 11%. Dual infection was identified in the final specimens of five subjects, after as much as 18 months follow-up, while for the remaining five subjects, dual infection was identified in interim specimens within an average of 10 months follow-up. Analysis of samples obtained after dual infection from each of these latter five subjects revealed two patterns: reversion to initial strain, or replacement of initial strain. Four subjects were dually-infected with HIV-1 strains of the same subtype, while 6 were infected with different subtypes. CONCLUSIONS: The high prevalence of recombinant HIV-1 strains in Cameroon may in part be explained by the high frequency of dual infection. In this genetically-diverse HIV-1 milieu, dual infections and the recombinant viruses they generate are strongly driving viral evolution, complicating vaccine strategies.

A heteroduplex assay for the rapid detection of dual Human Immunodeficiency Virus Type 1 infections.

The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.

Utility of the heteroduplex assay (HDA) as a simple and cost-effective tool for the identification of HIV type 1 dual infections in resource-limited settings.

The predominance of unique recombinant forms (URFs) of HIV-1 in Cameroon suggests that dual infection, the concomitant or sequential infection with genetically distinct HIV-1 strains, occurs frequently in this region; yet, identifying dual infection among large HIV cohorts in local, resource-limited settings is uncommon, since this generally relies on labor-intensive and costly sequencing methods. Consequently, there is a need to develop an effective, cost-efficient method appropriate to the developing world to identify these infections. In the present study, the heteroduplex assay (HDA) was used to verify dual or single infection status, as shown by traditional sequence analysis, for 15 longitudinally sampled study subjects from Cameroon. Heteroduplex formation, indicative of a dual infection, was identified for all five study subjects shown by sequence analysis to be dually infected. Conversely, heteroduplex formation was not detectable for all 10 HDA reactions of the singly infected study subjects. These results suggest that the HDA is a simple yet powerful and inexpensive tool for the detection of both intersubtype and intrasubtype dual infections, and that the HDA harbors significant potential for reliable, high-throughput screening for dual infection. As these infections and the recombinants they generate facilitate leaps in HIV-1 evolution, and may present major challenges for treatment and vaccine design, this assay will be critical for monitoring the continuing pandemic in regions of the world where HIV-1 viral diversity is broad.

Circulating recombinant form (CRF) 37_cpx: an old strain in Cameroon composed of diverse, genetically distant lineages of subtypes A and G.

HIV-1 in Cameroon is genetically diverse, but is predominated by the circulating recombinant form (CRF) 02_AG, which cocirculates among an array of other CRFs, unique recombinant forms (URFs), and all group M subtypes. In particular, our studies of HIV-1 diversity in the East Province found a high proportion of URFs and second generation recombinants (SGRs), suggesting this region of Cameroon may be a breading ground for new CRFs. Herein we present the full-length sequence analysis of one such CRF, composed primarily (66%) of unique, distant lineages of subtypes A and G in alternating regions throughout the genome. This CRF also combines segments in pol and env genes possessing intrasubtype distance (<15%) to the CRF01_AE and CRF02_AG radiations. The genomic composition of this strain comprising gene segments of subtypes A and G as well as CRF01_AE and CRF02_AG defines this strain as a circulating SGR (CSGR), and the 37th CRF to be identified. Furthermore, more than half of CRF19_cpx, a CRF identified in Cuba, clusters with CRF37_cpx, and the clear genetic distance among the viruses in this cluster suggests this strain has been in circulation since the early days of the epidemic. The genetically distant segments comprising CRF37_cpx, which were found to cluster outside the crown groups of previously described viruses, may represent a link to very rare or extinct strains, and, potentially, to understanding the evolutionary history of HIV-1 in this region.

Identification of a novel circulating recombinant form (CRF) 36_cpx in Cameroon that combines two CRFs (01_AE and 02_AG) with ancestral lineages of subtypes A and G.

An array of CRFs have been identified in Cameroon, the most notable being CRF02_AG. HIV-1 in the East Province of Cameroon is particularly diverse: in a recent study, we found a high proportion of unique recombinant forms (URFs). Herein we describe the analysis of the full-length sequences of two of these URFs, which, after preliminary analysis of gag, pol, and env fragments, appeared to be a novel CRF. This novel strain, CRF36_cpx, contains fragments that can be assigned to the CRF01_AE, CRF02_AG, and subtype A and G radiations. Forty percent of the genome can be classified as CRF02_AG, including regions in gag, pol, env, and the accessory genes. Twenty-seven percent is CRF01_AE, comprising the majority of gag, the beginning of env, and the end of env into the 3' LTR. Twenty percent of the genome can be assigned to subtype A, with segments in pol and env. The remaining 13% of the sequence is classifiable as subtype G, in pol and vpu. The subtype A and G lineages formed by the CRF36_cpx sequences are unique and appear ancestral in nature. CRF36_cpx is both the first to combine more than one CRF and the first to include fragments of CRF02_AG. The ancestral sequences present in CRF36_cpx represent a link to extinct strains, and, potentially, insight into the evolution of HIV-1.

Quasispecies analysis of novel HIV-1 recombinants of subtypes A and G reveals no similarity to the mosaic structure of CRF02_AG.

HIV-1 circulating recombinant form (CRF) 02_AG is responsible for greater than 65% of HIV-1 infections in Cameroon and is widespread across West and West-Central Africa. The parental subtypes A1 and G cocirculate in this part of Africa, and high rates of infection predispose to the generation of AG unique recombinant forms (URFs). Little is known as to whether A1 and G can recombine and thrive in vivo with breakpoints other than those characteristic of CRF02_AG. In this study, six unique recombinant viruses of subtypes A1 and G were identified in two individuals in Cameroon. A 1.5 kb fragment of the reverse transcriptase (RT) region of pol (HXB2 location 2,612-4,159) and the entire env gene (HXB2 location 6,202-9,096) were evaluated by phylogenetic and breakpoint analyses. Each URF was found to have breakpoints different than CRF02_AG, indicating that A and G gene segments are functionally compatible with more than one pattern of recombination. Furthermore, contemporaneous, cultured viruses from these individuals were analyzed, revealing different proportions of URFs compared to those found in plasma, possibly indicating compart mentalization and/or phenotypic variation among the URFs. CRF02_AG emerged from West-Central Africa to become a highly successful viral strain. As such, monitoring the spread of newly emerging AG recombinants is critical not only for understanding the epidemiology of HIV-1, but also in the design of future therapeutics and vaccines appropriate to this part of Africa, and globally.

Genetic analysis of HIV-1 strains in rural eastern Cameroon indicates the evolution of second-generation recombinants to circulating recombinant forms.

The HIV-1 genetic diversity in most parts of Cameroon is well described and shown to be very broad. However, little is known about the composition of the HIV-1 epidemic in the rural parts of eastern Cameroon. Therefore, we investigated 25 specimens from this region for their subtypes in gag, pol, and env gene fragments. Along with genetic material of subtypes A1, C, G, CRF01_AE, CRF02_AG, and CRF11_cpx, we also identified a large number (24%, 6/25) of distinct env sequences within the subtype A radiation. CRF02_AG was the predominant genetic form in all genes studied. Half of the specimens studied were considered "pure" based on concordant subtypes in the genes studied, whereas the other half were unique recombinant forms (URFs). Except for 1 URF, all were second-generation recombinants (SGRs), 90% of which contained genetic material of CRF02_AG in at least 1 gene. Notably, we identified individuals from 3 different villages infected with CRF01_AE(gag)CRF02_AG(pol)A(env) strains, which is indicative of the evolution of this URF to a circulating recombinant form (CRF). In addition, we identified a CRF02_AG(pol)C(env) recombinant infecting a man and a woman living in the same village, suggesting horizontal transmission of this recombinant. The current study emphasizes the power of HIV-1 recombination through the generation of SGRs and the evolution of URFs into CRFs. These findings suggest that, in a region where a predominant HIV-1 strain cocirculates among several subtypes, recombination could eventually decrease the proportion of this strain over time, such as CRF02_AG in Cameroon.

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form 02_AG (CRF02_AG) has a higher in vitro replicative capacity than its parental subtypes A and G.

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 02_AG is the predominant subtype in Cameroon, even more prevalent than the parental subtypes A and G. An important question that needs to be addressed is whether recombination in HIV-1 infection can lead to the emergence of viruses with biological advantages. The replicative capacity was investigated in peripheral blood mononuclear cells (PBMCs) of 13 R5-tropic primary HIV-1 isolates, including 5 CRF02_AG, 4 subtype A, and 4 subtype G viruses. HIV-1 subtype identity was defined by phylogeny either of the full-length genome or analysis of a combination of segments of the gag, pro, pol, and env genes followed by recombination breakpoint analysis. All viruses were grown on PBMCs for 11 days and culture supernatant was analyzed for reverse transcriptase (RT) activity and p24 production. On day 11 post-infection, CRF02_AG strains had a 1.4-1.9 times higher RT activity and reached a significantly higher level of p24 production than the parental subtypes A and G. Furthermore, the replication rate as measured by p24 production was 1.4 times higher for CRF02_AG strains compared to the subtypes A and G. This study suggests that the recombination event that led to CRF02_AG resulted in a variant with a better replicative capacity than its progenitors. This adaptation could contribute to the broader spread of HIV-1 CRF02_AG leading to its predominance in West Central Africa compared to the lower prevalence of its parental subtypes A and G.

Infection with HIV type 1 group M non-B subtypes in individuals living in New York City.

OBJECTIVE: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. DESIGN: From October 1999 through April 2003, HIV-1-seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. METHODS: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. RESULTS: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE-like (n = 7; 13%), CRF02_AG-like (n = 19; 34%), an intersubtype recombinant form G/A (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). CONCLUSION: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.