We hypothesized that the median CD4 cell count at ART initiation and TB case finding over the years would have increased, and that an associated decrease in mortality would have occurred. The Adult Infectious Diseases Clinic (AIDC) at the Infectious Diseases Institute (IDI), at IWR-1 clinical trial the Makerere University College of Health Sciences in Kampala, Uganda, has provided out-patient HIV care since its inception in 2002. Treatment is based on the national guidelines of the Ugandan Ministry of Health, and consists of daily co-trimoxazole prophylaxis for all patients

irrespective of CD4 count, and ART initiation in those with a prior AIDS diagnosis (WHO stage IV disease) or a CD4 count <250 cells/μL [14, 15]. This CD4 count threshold was raised from <200 cells/μL in 2009. First-line ART comprises stavudine (d4T) or zidovudine (ZDV) in combination with lamivudine (3TC) plus a nonnucleoside reverse transcriptase inhibitor in standard doses [nevirapine (NVP) or efavirenz (EFV)]. The choice of ART is at the physician's discretion and is also dependent on availability. Screening for active opportunistic selleck chemicals llc infections including

TB takes place prior to ART initiation. Available investigations for TB include sputum microscopy, chest radiology, abdominal ultrasonography, and fine-needle lymph node aspiration for acid-fast bacilli microscopy and cytology. Diagnosis of TB is made on the basis of these investigations, but very often on presentation of symptoms only. Patients diagnosed with active TB are treated with standard WHO-recommended regimens [16]. A specialized outdoor TB/HIV clinic was set up on the IDI grounds in 2008, which centralized all TB and HIV care for both TB suspects and patients on TB treatment. Dedicated medical officers and nurse-counsellors were trained in diagnosis and management of the coinfection, and more systematic screening and follow-up were implemented. Scheduled clinic appointments take place every 4 weeks with monitoring of

clinical status and adherence. CD4 cell counts are performed every 6-phosphogluconolactonase 6 months using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA). Viral load monitoring is not routine and is only available for patients suspected of virological failure on clinical and immunological grounds. Patients requiring in-patient care are referred to Mulago Hospital, a tertiary care hospital in the same complex. All care at the IDI is free of charge. Data on clinical parameters, ART and adherence, WHO stage, toxicities and opportunistic infections are routinely collected into a database, to which laboratory data are added electronically. Pharmacy data on TB drug prescriptions were used to validate this database, as previously described [17].

To overexpress these proteins, salicylate (SAL) can be used to block the activity of MarR (Martin & Rosner, 1995) and paraquat (PQ) can oxidize and hence activate SoxR (Demple, 1996). Alternatively, 2,2′- or 4,4′-dipyridyl (DIP) enhances post-translational activation of Rob (Rosner et al., 2002). As a result of the homology in their DNA binding domains, these proteins activate overlapping regulons leading to two major phenotypes: (1) the superoxide resistance phenotype, which depends upon increasing the expression of the sodA, fpr, acnA, zwf, and fumC genes,

among others; and (2) the multiple antibiotic or multidrug resistance (MDR) phenotype, which mostly depends on activation of the acrAB, tolC, and micF genes (Pomposiello et al., 2001; Martin & Rosner, 2002). However, these activators CH5424802 differ in the extents to which they activate particular promoters, for example, SoxS activates fpr to a Adriamycin purchase much greater extent than MarA does. According to these differences, overexpression of SoxS leads to greater superoxide resistance than overexpression of MarA. The primary basis of these effects is because of small differences in the binding affinities of the proteins to the DNA, particularly to the binding sequences termed

soxbox, when SoxS is the primary activator, or marbox, when all three activators can bind and activate the downstream genes (Fawcett & Wolf, 1995; Martin et al., 2000; Martin & Rosner, 2011). Mutations within marR (leading to a lack of repressor function) and soxR (leading to a constitutively active state) have been found to overexpress the corresponding activators, MarA and SoxS, and hence show an MDR phenotype in addition to organic solvent tolerance associated with the overexpression of the efflux pump AcrAB/TolC (Oethinger et al., 1998; Kern et al., 2000; Koutsolioutsou et al., 2005). In a previous study of our group (Fabrega et al., 2010), the next differences in gene expression between an MDR

E. coli selected in vitro and its susceptible parental clinical isolate were analyzed. Several genes were found to be up-regulated in the resistant mutant, for example, soxS, marA, acrAB, and ompN, and a mutation within soxR, leading to a truncated form of the protein and thus to a constitutively active state, was detected as the most likely explanation for the MDR phenotype. This work has focused on the study of the increased expression of the ompN gene and its possible link with the resistance phenotype. OmpN, like OmpX and OmpW, is one of the minor porins present in E. coli that are poorly expressed and it is closely related to other quiescent porins such as the OmpS1 of Salmonella Typhi and OmpK36 of Klebsiella pneumonia. Moreover, it displays functional properties (single-channel conductance) that closely resemble those of the OmpC porin (Prilipov et al., 1998). However, the physiological role of OmpN is yet to be determined. The bacterial strains and plasmids used in this study are listed in Table 1.

1N, altered the

distribution of actin and 4.1N. In contrast, the KCC2-C568A mutant, which shows a reduced binding affinity to 4.1N, did not affect the cytoskeleton. Thus, we suggest that the interaction between KCC2 and 4.1N plays a key role in the induction of the developmental defects observed in the transgenic embryos. As KCC2-FL and KCC2-ΔNTD had an effect on migration of neural crest cells, we assessed whether ectopic expression could also affect neuronal migration in vitro. C17.2 HDAC inhibitors cancer cells were transfected with control, KCC2-FL, KCC2-ΔNTD and KCC2-C568A plasmids. After 48 h, a scratch was made through the cell layer and the cells were incubated in serum-reduced medium for 18 h to allow migration in the wound area. In control cultures, the wound area was invaded by a moderate number of cells (Fig. 9A). KCC2-FL (Fig. 9B) and KCC2-ΔNTD (Fig. 9C) transfections significantly reduced the number of migrating cells (73 and 72% of control; P = 0.016 and P = 0.011, respectively). Transfection with KCC2-C568A (Fig. 9D) did not affect the number of cells in the wound area (96% of control; P = 0.627). Thus, KCC2-FL and KCC2-ΔNTD perturbed migration of neuronal cells in vitro, similar to the effect on neural crest migration in vivo. Our work shows that ectopic expression of KCC2 in mouse embryos leads to disturbances in the actin cytoskeleton, which in turn interferes

with neuronal differentiation and migration. The results are consistent with a structural role for KCC2 during early neuronal development that is not dependent selleck chemicals llc on the ion transport function of KCC2. In several parts of the central nervous system, such as the spinal cord (Delpy et al., 2008) and brainstem

(Balakrishnan et al., 2003; Blaesse et al., 2006), KCC2 is expressed before the onset of functional Cl− extrusion. Moreover, the levels Endonuclease of KCC2 expression in the auditory brainstem do not change at the periods of the hyperpolarizing EGABA shift (Balakrishnan et al., 2003; Vale et al., 2005). It has been suggested that the early expressed protein is inactive and requires regulation of its localization, state of phosphorylation, or oligomerization for functional activation (Vale et al., 2005; Blaesse et al., 2006; Lee et al., 2007; Hartmann et al., 2009). KCC2 shows a high level of expression in the proximity of excitatory synapses and within dendritic spines (Gulyas et al., 2001) and, more recently, is has been shown that KCC2 promotes the development of spines through interaction with the cytoskeleton-associated protein 4.1N (Li et al., 2007). Thus, KCC2 has a morphogenic role that is independent of its ion transport function. This morphogenic role may explain the early presence of KCC2 prior to the hyperpolarizing EGABA shift. The present results show that KCC2 is already endogenously expressed at E9.5 in neuronal cells of mouse embryos. This is earlier than previously shown time points for KCC2 expression (Li et al.

The interaction was labile to oxidants, such as diamide Bcl 2 inhibitor and menadione. Based on these data, NCgl0899 was named spiA (stress protein interacting with WhcA). Physical association and dissociation of the purified His6–WhcA and GST–SpiA fusion proteins, as assayed by in vitro pull-down experiments, were consistent with in vivo results. These data indicated that the

interaction between WhcA and SpiA is not only specific but also modulated by the redox status of the cell and the functionality of the WhcA protein is probably modulated by the SpiA protein. Corynebacterium glutamicum is a Gram-positive bacteria that belongs to the order Actinomycetales, which also includes the genera Mycobacterium and Streptomyces (Ventura et al., 2007). Corynebacterium glutamicum is a remarkable organism and is capable of producing a variety of amino acids and nucleotides in large quantities (Leuchtenberger et al., 2005). Because of the industrial importance of this organism, its relevant genetic and biochemical features have been extensively characterized. Accordingly, strategies that C. glutamicum cells adopt in response to cellular stresses have attracted scientific interests in recent years. WhiB-like genes are a class of genes that perform diverse cellular processes, such as cell division, differentiation, pathogenesis, starvation survival, and stress

response (Gomez, 2000; Steyn et al., 2002; FXR agonist Kim et al., 2005; Geiman et al., 2006; Raghunand & Bishai, 2006; Singh et al., 2007; Choi et al., 2009). The whiB gene, which was originally identified and characterized in Streptomyces coelicolor, is a developmental regulatory gene that is essential for the sporulation of aerial hyphae (Davis & Chater, 1992). The whiB homologues are only found in the order Actinomycetales. Seven whiB homologues have been identified in the Mycobacterium tuberculosis

genome and at least six are present in S. coelicolor (Soliveri et al., 2000), whereas only four are found in C. glutamicum (Kim et al., 2005). The WhiB-like Liothyronine Sodium proteins have four conserved cysteine residues that bind to a redox-sensitive Fe–S cluster (Jakimowicz et al., 2005; Alam et al., 2007; Singh et al., 2007; Crack et al., 2009; Smith et al., 2010), which plays a critical role in controlling protein function. In general, the cluster loss reaction followed by oxidation of the coordinating cysteine thiols that form disulfide bridges is important for activity. For example, S. coelicolor WhiD loses its Fe–S cluster upon exposure to oxygen (O2) and the apo-WhiD may play important roles in cell physiology (Crack et al., 2009). Some WhiB-like proteins may function as transcription factors, as suggested by the presence of predicted helix–turn–helix DNA-binding motif. Recently, the M. tuberculosis WhiB1 protein in its apo-form was shown to have DNA-binding activity (Smith et al., 2010).

44; 95% AZD6738 confidence interval (CI) 1.24−9.57; P < 0.05] and ‘other’ Black (born outside sub-Saharan Africa) ethnicity (AOR 4.63; 95% CI 1.06–20.11; P < 0.05). We also found an association between older age and decreased likelihood of lifetime IPV (AOR 0.92; 95% CI 0.86–0.97;

P < 0.05). Over half of the women in this study reported lifetime experience of IPV. We found associations between IPV and mental health problems, younger age and other Black ethnicity. In view of its high prevalence, we advocate greater awareness of IPV among HIV healthcare professionals and recommend universal screening. Intimate partner violence (IPV) is defined as physical, sexual or psychological harm by a current or former partner or spouse [1]. The World Health Organization's multi-country study found that lifetime prevalence of physical and/or sexual partner violence was between 15 and 71% [2]. IPV is estimated to affect 28% of women living in the UK in their lifetime [3]. The social, psychological and physical consequences of IPV are considerable and it has been shown to SB431542 ic50 have adverse effects on health in both the short and long term [4]. Women experiencing IPV are more likely to be in regular contact with healthcare professionals than

women who are not experiencing IPV, providing important opportunities to identify women and offer support [5]. This has led to the UK’s Department of Health recommending that all National Health Service (NHS) trusts work towards routinely asking women about their experiences of IPV in clinical

settings [6]. Women living with HIV are more likely to experience IPV than HIV-negative women [7-10]. IPV may predate the HIV diagnosis or follow as a consequence of it [11]. IPV is a risk factor for HIV acquisition, possibly as a result of nonconsensual sex or difficulties negotiating safer sex [12-15]. Furthermore, male perpetrators of IPV are more likely to have HIV or other sexually transmitted infections (STIs) than nonperpetrators [16, 17]. IPV is also a predictor of worse HIV outcomes [18]. It may impair a woman’s ability to disclose her HIV status to her partner [19, 20], and to make appropriate decisions about health, including attendance at clinic appointments [21, 22], adherence to medication [23], and abstaining from breastfeeding to prevent mother-to-child transmission [9]. Adenosine triphosphate In view of the recognized paucity of data on IPV in women living with HIV in the UK [24], we conducted a study of women attending the HIV out-patient department at the Homerton University Hospital. The hospital is in Hackney in East London, an area with significant socioeconomic deprivation [25] and where local lifetime prevalence of physical IPV is as high as 41% in women attending primary care [26]. Within our HIV clinic population there are high levels of social vulnerability [27] and a higher proportion of female patients than in many other UK centres.

g. transplantation) or high heterogeneity among the groups in a chronic disease category (i.e. autoimmune diseases, rare diseases and endocrine diseases). In 2007, the SMR was 8.8, indicating a probability of death in HIV-infected patients more than 8 times higher than that in the general

population. The 2006 SMR for HIV infection was similar. Regarding the association of HIV infection with chronic disease groups, the most relevant results were the following: a very strong association between HIV infection Selleckchem Gefitinib and chronic liver diseases (SHR>8), stable over the years sampled; In 2007, the average per capita cost of medical services in the general population was equal to €1069 (Table 2); there was a marked difference between people with chronic diseases (27% of the population), who represented an average per capita cost of €3018, TSA HDAC in vivo and patients without chronic diseases, for whom per capita spending was €340. For HIV-infected patients, the average per capita cost in the year 2007 was €9894; for this cost, HIV-infected patients ranked third after transplantation patients (€19 829) and those with renal insufficiency (€13 927). However, when population costs were considered, HIV infection ranked 12th out of

15 disease categories, with a total cost of €28 621 971 (range €663 289 797 for cardiovascular and cerebrovascular diseases to €18 328 024 for rare diseases). Two-thirds of the average per capita costs for HIV-infected however patients were attributable to drugs, especially antiretroviral drugs, which represented 63% of the total cost. As shown in Table 3, in the period under examination there

was an increase in per capita cost of 5.7% annually, with a sizable acceleration between 2005 and 2006 (+10%). The per capita cost for in-hospital care steadily decreased (−3.6% annually), while the cost for drugs steadily increased (+10.1% annually), with an especially large jump between 2005 and 2006, which could be attributed to a 20% increase in the cost of antiretroviral drugs. New cases had lower costs than prevalent cases, and over 50% of this difference could be attributed to the higher in-hospital care costs for HIV-infected patients that have been identified prior to 2003. Spending was strongly influenced by the presence of chronic diseases. For instance, in the year 2007, average per capita cost was €8104 for the 1972 HIV-infected patients without other chronic diseases, while it was €12 013 when AIDS-related and non-AIDS-related cancers were associated with HIV infection, €11 370 when it was combined with chronic liver diseases, and €9908 for HIV infection associated with cardiovascular and cerebrovascular diseases. Estimated medical costs for the 10 most frequent chronic diseases in HIV-infected patients and for HIV infection alone in the years examined are shown in Table 4.

Although the serotypes and promoters we tested expressed strongly in cortical pyramidal neurons, cerebellar Purkinje cells, olfactory granule neurons, and striatal interneurons, they produced very little expression in cortical interneurons and granule neurons of the dentate gyrus and cerebellum. Expression in these cell types might be attained using different serotypes and promoters, but must be tested empirically. Finally, there is a strict temporal window during which this technique can be used. Injections must be performed within the first Selleck GSK1120212 12–24 h after birth for AAV1, and within the first few days for AAV8. The timing of AAV injection may also limit which cell types can be transduced, as several neuronal populations

are generated after birth. After injection, however, expression of viral transgenes can be readily delayed

using temporal control elements such as Cre recombinase – estrogen receptor and tTA. By optimising its natural mosaic transduction pattern, we discovered that neonatal viral transgenesis opens a wide range of experimental opportunities that are not possible with existing Nivolumab supplier methods. Cell-autonomous and cell-extrinsic effects can now be readily distinguished. Purkinje neurons can now be easily manipulated and imaged in vivo. New constructs can be rapidly screened without germline transgenesis. The final advantage of the approach is the rising availability of compatible off-the-shelf viral preparations (e.g. Penn Vector Core and UNC Gene Therapy Center) and vectors (e.g. Addgene) that can be custom packaged into a variety of serotypes. These

resources for viral manipulation complement Phenylethanolamine N-methyltransferase a growing community of mouse repositories where newly characterised mutant strains can be purchased online (e.g. Jackson Laboratories, MMRRC, GENSAT, EMMA). As both the pattern and expression level of viral-delivered transgenes can depend on a number of factors including the transgene itself, construct design (i.e. promoters and enhancers), capsid serotype, quality of the viral preparation, and viral titer, each new application will require some optimisation. However, the richness of viral manipulation and the rate at which it has recently advanced suggest that, with additional experimentation, a wide range of cell type specificities and novel applications are within reach. We thank Kazuhiro Oka and the Baylor College of Medicine Viral Vector Core for AAV production, Anna Gumpel, Carolyn Allen, Yuanyuan Zhang, and Bryan Song for mouse care, Bernard Lee and Bernard Kuecking from Zeiss for microscope support, Ben Arenkiel for sharing the EF1α-iCre-2A-tdTomato AAV vector, and Roy Sillitoe and Ben Arenkiel for helpful comments on the manuscript. Grant support was from American Health Assistance Foundation Alzheimer’s Disease Research Grant A2010097, National Institute of Aging R21 AG038856, and National Institutes of Health Office of the Director New Innovator Award DP2 OD001734. None.

Altered fat distribution and greater central adiposity were associated with detectable virus but not ART class(es) received. Poor growth is a common manifestation of HIV infection in children [1–5], the pathophysiology of which remains poorly understood. The importance of growth is underscored by the finding that height growth velocity predicts survival, regardless of plasma viral load [HIV-1 RNA (VL)], age and CD4 cell count [6]. The relationships among growth, VL, immune function and antiretroviral therapy (ART) remain unclear. Conflicting data exist from both pre- and post-highly active antiretroviral therapy (HAART) eras [6–13] about whether VL is associated with growth. Most, but not all [11–15], reports

PD-0332991 datasheet of children on protease inhibitor (PI) therapy note improved linear and ponderal growth. Some data suggest an association

with VL that is not independent of immune function [10]. It is still unclear whether improved growth sometimes seen with treatment is primarily a result of immune restoration, improved viral control or yet another mechanism. HIV infection and/or ART may also alter body composition, measurement of which may help differentiate starvation (preferential loss http://www.selleckchem.com/products/BIBF1120.html of fat resulting from inadequate energy intake) from cachexia [loss of lean body mass (LBM)], generally accepted to be cytokine mediated. Data are conflicting about preservation of LBM in HIV-infected children [2,16]. Altered fat tuclazepam distribution in HIV-infected persons, particularly those on ART, may also occur [17]. In particular, increased central adiposity has been reported in both HIV-infected adults and children [17,18], and is of concern because of the known association with cardiovascular morbidities [19]. Although limited information is available on associations and predictors of body composition and fat distribution in prepubertal HIV-infected children, exposure to PIs is frequently noted in association with lipodystrophy [18, 20–22]. Data regarding association with disease measures such as VL and CD4 percentage,

however, are conflicting [20,21]. The objectives of this study were (a) to describe growth and body composition changes in HIV-infected children over 48 weeks after beginning or changing ART; (b) to compare these changes in HIV-infected children to both US population-based data and data for matched, HIV-exposed, uninfected children; (c) to correlate growth and body composition changes with ART class(es) and changes in VL and CD4 cell percentage. We hypothesized that there is a clinically significant inverse correlation between changes in LBM and VL and a direct correlation between changes in LBM and CD4 cell percentage in children beginning or changing ART. We further hypothesized that there would be a greater increase in central adiposity in children who started therapy containing PIs compared with those who started non-PI regimens.

Altered fat distribution and greater central adiposity were associated with detectable virus but not ART class(es) received. Poor growth is a common manifestation of HIV infection in children [1–5], the pathophysiology of which remains poorly understood. The importance of growth is underscored by the finding that height growth velocity predicts survival, regardless of plasma viral load [HIV-1 RNA (VL)], age and CD4 cell count [6]. The relationships among growth, VL, immune function and antiretroviral therapy (ART) remain unclear. Conflicting data exist from both pre- and post-highly active antiretroviral therapy (HAART) eras [6–13] about whether VL is associated with growth. Most, but not all [11–15], reports

Akt inhibitor of children on protease inhibitor (PI) therapy note improved linear and ponderal growth. Some data suggest an association

with VL that is not independent of immune function [10]. It is still unclear whether improved growth sometimes seen with treatment is primarily a result of immune restoration, improved viral control or yet another mechanism. HIV infection and/or ART may also alter body composition, measurement of which may help differentiate starvation (preferential loss BIBW2992 ic50 of fat resulting from inadequate energy intake) from cachexia [loss of lean body mass (LBM)], generally accepted to be cytokine mediated. Data are conflicting about preservation of LBM in HIV-infected children [2,16]. Altered fat Protein kinase N1 distribution in HIV-infected persons, particularly those on ART, may also occur [17]. In particular, increased central adiposity has been reported in both HIV-infected adults and children [17,18], and is of concern because of the known association with cardiovascular morbidities [19]. Although limited information is available on associations and predictors of body composition and fat distribution in prepubertal HIV-infected children, exposure to PIs is frequently noted in association with lipodystrophy [18, 20–22]. Data regarding association with disease measures such as VL and CD4 percentage,

however, are conflicting [20,21]. The objectives of this study were (a) to describe growth and body composition changes in HIV-infected children over 48 weeks after beginning or changing ART; (b) to compare these changes in HIV-infected children to both US population-based data and data for matched, HIV-exposed, uninfected children; (c) to correlate growth and body composition changes with ART class(es) and changes in VL and CD4 cell percentage. We hypothesized that there is a clinically significant inverse correlation between changes in LBM and VL and a direct correlation between changes in LBM and CD4 cell percentage in children beginning or changing ART. We further hypothesized that there would be a greater increase in central adiposity in children who started therapy containing PIs compared with those who started non-PI regimens.

Data were collected using the SpectraSuite v1.6 software (Ocean Optics, Inc.). All measurements were conducted using

the U-MWIB filter cube at the same magnification (100× objective). Comparisons between samples were based on relative fluorescence intensity. Using the genomic DNA of C. velia, we successfully amplified SSU and ITS rRNA gene (GC content 46%). CV1 probe specific for C. velia (5′-CAA GAG AAT CGA GCA CGG-3′) was confirmed to be unique using ‘probeCheck’. There was no SSU rRNA gene sequence that would have one or two mismatches to CV1 probe. The closest hits were bacterial and archaeal sequences with three mismatches. The nearest confirmed eukaryote sequences are from Euglena spp. with four mismatches. Moreover, there were I-BET-762 purchase 15 mismatches or in-dels and 10 mismatches with the corresponding SSU rRNA gene of Symbiodinium sp. (Dinophyceae) and Vitrella brassicaformis (Chromerida) to the CV1 probe. Of the three hybridization protocols chosen from literature (see ‘Materials and methods’), the method (3) was the most effective for FISH detection of C. velia with the CV1 probe and was adopted as the protocol of choice for optimizations. Using the optimized paraformaldehyde/DTAB MG-132 purchase method, a clear difference between the intensity and distribution of green fluorescence was observed between the probed and un-probed slides. The

most effective hybridization duration for CV1 probe was 15 h at 48 °C, with a strong Demeclocycline FITC-related green fluorescence signal observed (Fig. 1). Hybridization of samples with CV1 probe for 4 h at 48 °C revealed weak FITC-related green fluorescence signal, while no green fluorescent signal was seen with 1 and 1.5 h of incubation. Using 15-h hybridization, 20–80% C. velia cells were positively labelled (Fig. 2). It was apparent in un-probed control slides that C. velia emits yellow autofluorescence (Figs 1 and 2). However, the signal obtained from probed cells designated as FISH-positive

showed a distinct difference in the distribution of fluorescence compared to that obtained from autofluorescence (Fig. 1). The yellow autofluorescence had an inconsistent, patchy appearance. Conversely, the cytoplasm of the probed C. velia cell was saturated with bright green FITC fluorescence. Additionally, a thin strip of yellow fluorescence was observed along the inner lining of the cell and was assumed to originate from the cell’s plastid. Using a spectrophotometer, we measured relative intensity of probed and un-probed C. velia fluorescence (Fig. 3). The CV1 probed C. velia emission spectrum showed a green peak consistent with green FITC fluorescence. The spectrum of un-probed C. velia demonstrated broad green/yellow autofluorescence (> 530 nm) corresponding to the observed yellow autofluorescence. Hybridizations of the mixed organism sample resulted in successful detection of C. velia cells by the CV1 probe among other free-living eukaryotes (Fig. 4).