Elucidating the Catalytic Subunit Composition of Distinct Proteasome Subtypes: A Crosslinking Approach Employing Bifunctional Activity‐Based Probes

In addition to two well‐recognized proteasome subtypes—constitutive proteasomes and immunoproteasomes—mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity‐based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.     

Mediated Electrochemistry to Mimic Biology's Oxidative Assembly of Functional Matrices

Biology uses diffusible oxidants to perform functions that range from signaling to matrix assembly, and these oxidation chemistries offer surprising selectivities. Here, it is reported that mediated electrochemistry can access the richness of such oxidation chemistries. Specifically, electrode‐imposed voltage inputs are used to locally generate oxidized mediators that can diffuse into polymer solutions and induce the formation of covalent bonds for the deposition and functionalization of hydrogels at the electrode surface. Depending on the mediator's redox potential (E⁰), it is possible to “gate” the voltage inputs to target specific residues (e.g., thiols or amines) and oxidation chemistries. Further, mediators of varying E⁰ offer different reactivities and thus allow control of reaction‐diffusion rates to modulate the hydrogel's crosslink density and mechanical properties. Importantly, this mediated oxidation can be performed under physiologically relevant conditions to preserve labile biological functionalities (e.g., cell viability and protein function). Finally, it is demonstrated that protein fusion tags can be engineered to have “targetable” amino acid residues that enable protein function to be oxidatively conjugated to electrodeposited hydrogels. In summary, mediated electrochemistry can engage orthogonal oxidation chemistries to create functionalized matrices and thus mediated electrochemistry should add important capabilities to the electrofabrication toolbox.     

Milk- and starter-feeding strategies to reduce cross sucking in pair-housed calves in outdoor hutches

Social housing of preweaned calves can benefit their welfare, but housing and cross sucking are potential barriers to adoption for farmers. For farms using outdoor hutches, an option is to pair adjacent hutches with a shared fence. Our objective was to investigate milk- or starter-feeding strategies to mitigate cross sucking in this system. Holstein heifers were housed in pairs (n = 32 pairs) and divided into 4 treatments (n = 8 pairs each) in a 2 × 2 factorial design: milk in an open bucket with starter in only a bucket (OB-SB), milk in an open bucket and starter in both a bucket and a specialized teat bottle (Braden bottle; OB-BB), milk in a slow-flow teat bucket with starter in only a bucket (TB-SB), or milk in a slow-flow teat bucket and starter in both a bucket and a specialized teat bottle (TB-BB). When starter was first offered (d 6 ± 1 of life, mean ± SD), calf latency to approach was recorded, averaged within pairs, and compared between starter treatments using a linear mixed model with fixed effect of treatment and random effect of pair within treatment. Calves were initially bottle fed; milk treatments began on d 14 ± 1 of life and ended when calves were completely weaned (d 53 ± 1). Calves were observed for behaviors such as drinking milk and cross sucking twice weekly for 30 min during the afternoon milk meal using continuous video, with values averaged within pairs. Linear mixed models were run separately before (wk 3–6) and after (wk 7–8) weaning, with fixed effects of milk- and starter-feeding treatments, week, and the 2- and 3-way interactions, with week as repeated measure and pair as subject. Pairs with Braden bottles and buckets approached starter sooner than those with only buckets (Braden bottles vs. no Braden bottles: 13.1 ± 6.1 vs. 33.2 ± 6.1 min, LSM ± SEM). Before weaning, pairs with open buckets for both milk and starter cross sucked for at least twice as long (OB-SB: 2.9 ± 0.3 min) as all other treatments (OB-BB: 1.5 ± 0.3 min; TB-SB: 0.4 ± 0.3 min; TB-BB: 0.5 ± 0.3 min). This pattern held during weaning, when cross sucking increased overall (OB-SB: 3.9 ± 0.4 min; OB-BB: 1.8 ± 0.4 min; TB-SB: 0.9 ± 0.4 min; TB-BB: 1.6 ± 0.4 min). Regardless of starter treatment, calves spent less time cross sucking when fed milk in teat buckets, which extended the milk meal relative to open buckets (teat bucket vs. open bucket: preweaning = 7.0 ± 0.2 vs. 1.6 ± 0.2 min; weaning = 3.0 ± 0.1 vs. 0.6 ± 0.1 min). When calves are fed milk in open buckets, a novel option for reducing cross sucking is to provide starter through a specialized bottle. Nonetheless, providing milk in slow-flow teat buckets had the greatest effect on reducing cross sucking by directing suckling to the teat instead of another calf or pen objects.     

Sources and temporal dynamics of arsenic in a New Jersey watershed, USA

We examined potential sources and the temporal dynamics of arsenic (As) in the slightly alkaline waters of the Wallkill River, northwestern New Jersey, where violations of water-quality standards have occurred. The study design included synoptic sampling of stream water and bed sediments in tributaries and the mainstem, hyporheic-zone/ground water on the mainstem, and seasonal and diurnal sampling of water at selected mainstem sites. The river valley is bordered by gneiss and granite highlands and shale lowlands and underlain by glacial deposits over faulted dolomites and the Franklin Marble. Ore bodies in the Marble, which have been mined for rare Zn ore minerals, also contain As minerals. Tributaries, which drain predominantly forested and agricultural land, contributed relatively little As to the river. The highest concentrations of As (up to 34 mug/L) emanated from the outlet of man-made Lake Mohawk at the river's headwaters; these inputs varied substantially with season--high during warm months, low during cold months, apparently because of biological activity in the lake. Dissolved As concentrations were lower (3.3 microg/L) in river water than those in ground water discharging into the riverbed (22 microg/L) near the now-closed Franklin Mine. High total As concentrations (100-190 mg/kg) on the <0.63 microm fraction of bed sediments near the mine apparently result from sorption of the As in the ground-water discharge as well as from the As minerals in the streambed. As concentrations in river water were diluted during high stream flow in fall, winter and spring, and concentrated during low flow in summer. In unfiltered samples from a wetlands site, diurnal cycles in trace-element concentrations occurred; As concentrations appeared to peak during late afternoon as pH increased, but Fe, Mn, and Zn concentrations peaked shortly after midnight. The temporal variability of As and its presence at elevated concentrations in ground water and sediments as well as streamwater demonstrate the importance of (1) sampling a variety of media and (2) determining the time scales of As variability to fully characterize its passage through a river system.     

Effect of watershed parameters on mercury distribution in different environmental compartments in the Mobile Alabama River Basin, USA

Total mercury (THg) and mono-methylmercury (MeHg) levels in water, sediment, and largemouth bass (LMB) (Micropterus salmoides) were investigated at 52 sites draining contrasting land use/land cover and habitat types within the Mobile Alabama River Basin (MARB). Aqueous THg was positively associated with iron-rich suspended particles and highest in catchments impacted by agriculture. Sediment THg was positively associated with sediment organic mater and iron content, with the highest levels observed in smaller catchments influenced by wetlands, followed by those impacted by agriculture or mixed forest, agriculture, and wetlands. The lowest sediment THg levels were observed in main river channels, except for reaches impacted by coal mining. Sediment MeHg levels were a positive function of sediment THg and organic matter and aqueous nutrient levels. The highest levels occurred in agricultural catchments and those impacted by elevated sulfate levels associated with coal mining. Aqueous MeHg concentrations in main river channels were as high as those in smaller catchments impacted by agriculture or wetlands, suggesting these areas were sources to rivers. Elevated Hg levels in some LMB were observed across all types of land use and land cover, but factors such as shallow water depth, larger wetland catchment surface area, low aqueous potassium levels, and higher Chl a concentrations were associated with higher Hg burdens, particularly in the Coastal Plain province. It is suggested that the observed large variability in LMB Hg burdens is linked to fish displacement by anglers, differences in food web structure, and sediment biogeochemistry, with surficial sediment iron oxides buffering the flux of MeHg from sediments to deeper water pelagic food webs.     

An Expanded Set of Fluorogenic Sulfatase Activity Probes

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3‐O‐methylfluorescein‐sulfate and resorufin‐sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species‐specific sulfatase banding patterns in a gel‐resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.     

Evaluating interfaces with children

Children are very different from adults—they have developing motor skills, limited reach, short attention spans, limited exposure to traditional user interfaces and social protocols. How do we evaluate user interfaces for children? In this paper, we present four evaluative techniques that we have successfully used with children.