Molecular paleontology: a biochemical model of the ancestral ribosome

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.     

Live and dead GFP-tagged bacteria showed indistinguishable fluorescence in Caenorhabditis elegans gut

Caenorhabditis elegans has been used for studying host-pathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.     

Transient Responses of Cell Turgor and Growth of Maize Roots as Affected by Changes in Water Potential

Transient responses of cell turgor (P) and root elongation to changes in water potential were measured in maize (Zea mays L.) to evaluate mechanisms of adaptation to water stress. Changes of water potential were induced by exposing roots to solutions of KCl and mannitol (osmotic pressure about 0.3 MPa). Prior to a treatment, root elongation was about 1.2 mm h-1 and P was about 0.67 MPa across the cortex of the expansion zone (3-10 mm behind the root tip). Upon addition of an osmoticum, P decreased rapidly and growth stopped completely at pressure below approximately 0.6 MPa, which indicated that the yield threshold (Ytrans,1) was just below the initial turgor. Turgor recovered partly within the next 30 min and reached a new steady value at about 0.53 MPa. The root continued to elongate as soon as P rose above a new threshold (Ytrans,2) of about 0.45 MPa. The time between Ytrans,1 and Ytrans,2 was about 10 min. During this transition turgor gradients of as much as 0.15 MPa were measured across the cortex. They resulted from a faster rate of turgor recovery of cells deeper inside the tissue compared with cells near the root periphery. Presumably, the phloem was the source of the compounds for the osmotic adjustment. Turgor recovery was restricted to the expansion zone, as was confirmed by measurements of pressure kinetics in mature root tissue. Withdrawal of the osmoticum caused an enormous transient increase of elongation, which was related to only a small initial increase of P. Throughout the experiment, the relationship between root elongation rate and turgor was nonlinear. Consequently, when Y were calculated from steady-state conditions of P and root elongation before and after the osmotic treatment, Yss was only 0.21 MPa and significantly smaller compared with the values obtained from direct measurements (0.42-0.64 MPa). Thus, we strongly emphasize the need for measurements of short-term responses of elongation and turgor to determine cell wall mechanics appropriately. Our results indicate that the rate of solute flow into the growth zone could become rate-limiting for cell expansion under conditions of mild water stress.     

Seed dormancy in Avena fatua: Interacting effects of nitrate, water and seed coat injury

In experiments conducted under controlled conditions. KNO₃ (50 or 100 m M ) promoted germination of a dormant strain (AN 474) of Avena fatua when either one or two holes were pierced in the lower (adaxial) surface of the caryopsis in contact with the nitrate solution. Germination was increased by increasing either the KNO₃ concentration or the number of holes in the seed coat. The germination response induced by the application of water to a hole pierced in the upper surface of the caryopsis was. increased by pre-treatment of the intact caryopsis with KNO₃. Treatment with either 50 or 100 m M KNO₃ caused a transient reduction in embryo water content of intact cary-opses, but increased the nitrate and amino- N content of pierced caryopses prior to germination. Supplying a 100 m M solution of KNO₃ to pierced caryopses reduced the total water potential and osmotic potential of the embryo, and increased its pressure potential by the same amount as an equimolar solution of KC1; however, while both treatments promoted germination, the KNO₃ induced more rapid germination than the KCI. Both treatments also increased the K⁺ content of the embryo, the KNO₃ again having the greater effect. These results are consistent with the hypothesis, based on our previous investigations, that KNO₃ promotes germination of dormant caryopses by accumulating in the embryo where it acts osmotically to increase water uptake. It is also postulated, that, in contrast to KCI, KNG₃ may combine an osmotic effect on water uptake with a nutritional effect on protein synthesis.     

Tillers Do Not Influence Phytotoxicity of Imazamethabenz in Wild Oat (Avena fatua)

The response of wild oat to imazamethabenz varies with the growth stage, but the role of tillers in this regard is unclear. Removal of tillers at the three-leaf stage before spraying with imazamethabenz did not significantly affect the total shoot fresh weight measured 3 weeks later. The leaf area and dry weight of intact plants at the three-leaf stage were 17–21% greater than for plants with coleoptilar and first leaf main shoot tillers (T0 and T1) removed. The greater leaf area may have increased herbicide interception per plant. Similar fresh weight reductions in main shoot, total tillers, and total shoots were found whether imazamethabenz was applied to the plant at the two-leaf without tillers or the three-leaf with two tillers stage. Imazamethabenz applied only to the main shoot reduced total shoot dry weight more than an equivalent amount of imazamethabenz applied only to tiller T1 or applied over the whole shoot. Imazamethabenz had the least inhibitory effect on whole plant growth when applied only to T1. When ¹⁴C-herbicide was applied to the first main shoot leaf of plants at the three-leaf stage with two tillers, the ¹⁴C translocated 38% to roots, 33% to the main shoot, and nearly 30% to all tillers. When ¹⁴C-herbicide was applied to the first leaf of T1 then the ¹⁴C translocated 50% to T1, 25% to the main shoot, 20% to roots, and 5% to all other tillers. The translocation pattern and fresh weight values suggested that the presence of early tillers during herbicide application neither increased nor decreased imazamethabenz efficacy in wild oat. Received June 4, 1997; accepted June 5, 1997     

Efficient assembly of rat hepatocyte spheroids for tissue engineering applications

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to cells cultured as monolayers. An efficient method of forming spheroids in spinner vessels is described. Within 24 h after inoculation, greater than 80% of inoculated cells formed spheroids. This efficiency was significantly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O(2)/cell/h, the oxygen supply is critical and should be monitored for successful formation. Throughout a 6-day culture period, spheroids assembled in spinner cultures maintained a high viability and produced albumin and urea at constant rates. Transmission electron microscopy indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channels were observed in the interior of spheroids and appeared to access the exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as well as similar morphology and ultrastructure compared to spheroids formed in stationary petri dishes. Hepatocytes cultured as spheroids are potentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism. (c) 1996 John Wiley & Sons, Inc.     

Heteroaryl and cycloalkyl sulfonamide hydroxamic acid inhibitors of matrix metalloproteinases

Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.     

Differential effects of Matrigel and its components on functional activity of CFTR and ENaC in mouse endometrial epithelial cells

Our previous studies have observed an effect of Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, on the expression of ion channels in mouse endometrial epithelia; namely the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-)channel, and the epithelial sodium channel (ENaC). The present study further investigated the effects of Matrigel and its individual components on the functional expression of CFTR and ENaC using the short-circuit current (Isc) technique. The results showed that different components of Matrigel, namely growth factors, laminin and collagen, had differential effects on the functional activity of the two ion channels in murine endometrial epithelium. The information obtained may be useful for designing future in vitro culture models to investigate the functional roles of these ion channels in the endometrium.     

Laboratory Evaluation of the Alere q Point-of-Care System for Early Infant HIV Diagnosis

Introduction

Early infant diagnosis (EID) and prompt linkage to care are critical to minimise the high morbidity and mortality associated with infant HIV infection. Attrition in the “EID cascade” is common; however, point-of-care (POC) EID assays with same-day result could facilitate prompt linkage of HIV-infected infant to treatment. Despite a number of POC EID assays in development, few have been independently evaluated and data on new technologies are urgently needed to inform policy.

Methods

We compared Alere q 1/2 Detect POC system laboratory test characteristics with the local standard of care (SOC), Roche CAP/CTM HIV-1 qualitative PCR in an independent laboratory-based evaluation in Cape Town, South Africa. Routinely EID samples collected between November 2013 and September 2014 were each tested by both SOC and POC systems. Repeat testing was done to troubleshoot any discrepancy between POC and SOC results.

Results

Overall, 1098 children with a median age of 47 days (IQR, 42–117) were included. Birth PCR (age <7 days) comprised of 8% (n = 92) tests while 56% (n = 620) of children tested as part of routine EID (ages 6–14 weeks). In the overall direct comparison, Alere q Detect achieved sensitivity of 95.5% (95% CI, 91.7–97.9%) and a specificity of 99.8% (95% CI, 99.1–100%). Following repeat testing of discordant samples and exclusion of any inconclusive results, the POC assay sensitivity and specificity were 96.9% (95% CI 93.4–98.9%) and 100% (lower 95% CI 98%) respectively. Among birth PCR tests the POC assay had slightly lower sensitivity (93.3% vs 96.5% in routine EID) and higher assay error rate (10% vs 5% in samples of older children, p = 0.04).

Conclusion

Our results indicate this POC assay performs well for EID in the laboratory. The high specificity and thus high positive predictive value would suggest a positive POC result may be adequate for immediate infant ART initiation. While POC testing for EID may have particular utility for birth testing at delivery facilities, the lower sensitivity and error rate requires further attention, as does field implementation of POC EID technologies in other clinical care settings.