Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Some biological features of Saprolegnia parasitica Coker - cause of fish dermatomycosis. [Translation from: Pervaya nauchnaya konferentsiya molodykh Uchenykh Biologov (Tezisy Dokladov) pp.84-86. Kiev, A.N.Ukr. SSR., 1964]

Parasitic and infectious diseases of fish, of wide distribution in fish-rearing ponds, retard to a significant extent the development of fish culture in the Ukraine. One of the diseases of fish attracting attention in connection with the general distribution of its causative agent, the fungus Saprolegnia parasitica Coker, in water-bodies of various types, appears to be dermatomycosis. The aim of this investigation is to study the conditions favouring the development of S. parasitica. Among the studied factors were water temperature and oxygen content.

On an embedding property of generalized Carter subgroups

If E and F are saturated formations, we say that E is strongly contained in F if for any solvable group G with E-subgroup, E, and F-subgroup, F, some conjugate of E is contained in F. In this paper, we investigate the problem of finding the formations which strongly contain a fixed saturated formation E.

Our main results are restricted to formations, E, such that E = {G|G/F(G) ϵT}, where T is a non-empty formation of solvable groups, and F(G) is the Fitting subgroup of G. If T consists only of the identity, then E=N, the class of nilpotent groups, and for any solvable group, G, the N-subgroups of G are the Carter subgroups of G.

We give a characterization of strong containment which depends only on the formations E, and F. From this characterization, we prove:

If T is a non-empty formation of solvable groups, E = {G|G/F(G) ϵT}, and E is strongly contained in F, then

(1) there is a formation V such that F = {G|G/F(G) ϵV}.

(2) If for each prime p, we assume that T does not contain the class, S_p’, of all solvable p’-groups, then either E = F, or F contains all solvable groups.

This solves the problem for the Carter subgroups.

We prove the following result to show that the hypothesis of (2) is not redundant:

If R = {G|G/F(G) ϵS_r’}, then there are infinitely many formations which strongly contain R.

Factors influencing the start of development in Daphnia pulex winter eggs [Translation from: Biological Reviews Vol. 13, 24-26, 1951]

The winter eggs of Daphnia pulex, after passing safely through the winter , develop and hatch in the spring, multiplying by themselves, while some males emerging among them with the changes in environment produce fertile eggs, which are universally known as winter eggs . This study researches the factors governing the development of winter eggs through experiments.

An in vitro biomolecular breadboard for prototyping synthetic biological circuits

Biomolecular circuit engineering is critical for implementing complex functions in vivo, and is a baseline method in the synthetic biology space. However, current methods for conducting biomolecular circuit engineering are time-consuming and tedious. A complete design-build-test cycle typically takes weeks' to months' time due to the lack of an intermediary between design ex vivo and testing in vivo. In this work, we explore the development and application of a "biomolecular breadboard" composed of an in-vitro transcription-translation (TX-TL) lysate to rapidly speed up the engineering design-build-test cycle. We first developed protocols for creating and using lysates for conducting biological circuit design. By doing so we simplified the existing technology to an affordable ($0.03/uL) and easy to use three-tube reagent system. We then developed tools to accelerate circuit design by allowing for linear DNA use in lieu of plasmid DNA, and by utilizing principles of modular assembly. This allowed the design-build-test cycle to be reduced to under a business day. We then characterized protein degradation dynamics in the breadboard to aid to implementing complex circuits. Finally, we demonstrated that the breadboard could be applied to engineer complex synthetic circuits in vitro and in vivo. Specifically, we utilized our understanding of linear DNA prototyping, modular assembly, and protein degradation dynamics to characterize the repressilator oscillator and to prototype novel three- and five-node negative feedback oscillators both in vitro and in vivo. We therefore believe the biomolecular breadboard has wide application for acting as an intermediary for biological circuit engineering.

Biological surveillance of chalk-streams

An article reviewing the methods of biological surveillance of chalk-streams developed and commonly used at that time, with a focus on their application to the River Frome catchment in Dorset. In evaluating the surveillance methods, the author looks at sampling methods (including cores and kick-sampling), the level of identification of macroinvertebrates, and temporal and spatial variations. Responses of indices to organic pollution are also discussed. A number of accompanying figures are also included.

Biological environments of larger UK rivers: progress report 1987

This interim progress report for the 9 months from January 1987 to September 1987 aims to provide insights into the mechanisms by which populations of particles (in this instance, phytoplankton) behave in relation to fluvial flow and, thus, to better model the dispersive properties of rivers and the ecological principles governing the distribution of potamoplankton generally. The author has been able to show with dye-tracers that significant water retention in pool reaches occurs within the range of (low) discharges obtaining, in accord with the Aggregated Dead Zone model and to an extent comparable with streams and small rivers investigated previously.

The first fifty years of the Freshwater Biological Association

A brief history of the Freshwater Biological Association is given in this article. The association has been in existence for fifty years, having been founded in 1929.

The Freshwater Biological Association and the International Biological Programme (1961-1976)

The involvement of the FBA in the primary productivity program is reviewed.

Biological and climatic influences on the dace Leuciscus leuciscus in a southern chalk-stream

The dace, Leuciscus leuciscus (L.) is an important cyprinid in terms of population biomass in chalk streams of southern England. Dace recruitment has been shown to vary widely from year to year and it is thought that this variation is largely as a result of the influence of abiotic factors, chiefly water temperature. From 1968 to 1981 there was a thirteen-fold difference in the year class structure index between the minimum index (0.25 in 1972) and the maximum (3.21 in 1976). The problems of such variation, especially those that could ensue from a succession of poor year-classes, are offset by the spread of reproductive effort by each female over several years.

Bassenthwaite Lake: a general assessment of environmental and biological features and their susceptibility to change

Bassenthwaite (Lake) is one of the larger Cumbrian lakes, certainly one of the most distinctive, and of considerable conservation and amenity value. Although its shores lack sizeable settlements, its main inflow receives sewage effluent from a major tourist centre (Keswick) and is subject to episodic floods. These influences, the growing development of leisure activities at the lake (e.g. sailing, time-share units), and recent road-construction, have led to past appraisals of ecological impacts and lake management. The lake has not been the subject of intense and long-term ecological study, but much scattered information exists that is relevant to future management decisions. In the present Report, commissioned by North West Water, such information - published and unpublished - is surveyed. Especial attention is given to evidence bearing on susceptibility to change, affecting the lake environment and its biota or species of conservation interest. Extensive use has been made of the results of a recent (1986-7) seasonal survey by the FBA.

A general assessment of environmental and biological features of Windermere and their susceptibility to change

Bassenthwaite (Lake) is one of the larger Cumbrian lakes, certainly one of the most distinctive, and of considerable conservation and amenity value. Although its shores lack sizeable settlements, its main inflow receives sewage effluent from a major tourist centre (Keswick) and is subject to episodic floods. These influences, the growing development of leisure activities at the lake (e.g. sailing, time-share units), and recent road-construction, have led to past appraisals of ecological impacts and lake management. The lake has not been the subject of intense and long-term ecological study, but much scattered information exists that is relevant to future management decisions. In the present Report, commissioned by North West Water, such information - published and unpublished - is surveyed. Especial attention is given to evidence bearing on susceptibility to change, affecting the lake environment and its biota or species of conservation interest. Extensive use has been made of the results of a recent (1986-7) seasonal survey by the FBA.

Description of sampling stations, methods of benthic sampling and biological water quality assessment; with some consideration of the influence of sample variation on the assessment values obtained

The first bilateral study of methods of biological sampling and biological methods of water quality assessment took place during June 1977 on selected sampling sites in the catchment of the River Trent (UK). The study was arranged in accordance with the protocol established by the joint working group responsible for the Anglo-Soviet Environmental Agreement. The main purpose of the bilateral study in Nottingham was for some of the methods of sampling and biological assessment used by UK biologists to be demonstrated to their Soviet counterparts and for the Soviet biologists to have the opportunity to test these methods at first hand in order to judge the potential of any of these methods for use within the Soviet Union. This paper is concerned with the nine river stations in the Trent catchment.

Corroborative studies on the effects of sample variation on three macroinvertebrate biological assessment methods at different water quality states

In recent collaborative biological sampling exercises organised by the Nottingham Regional Laboratory of the Severn-Trent Water Authority, the effect of handnet sampling variation on the quality and usefulness of the data obtained has been questioned, especially when this data is transcribed into one or more of the commonly used biological methods of water quality assessment. This study investigates if this effect is constant at sites with similar typography but differing water quality states when the sampling method is standardized and carried out by a single operator. An argument is made for the use of a lowest common denominator approach to give a more consistent result and obviate the effect of sampling variation on these biological assessment methods.