Scheduled and Breakthrough Opioid Use for Cancer Pain in an Inpatient Setting at a Tertiary Cancer Hospital.

Background: Our aim was to examine the frequency and prescription pattern of breakthrough (BTO) and scheduled (SCH) opioids and their ratio (BTO/SCH ratio) of use, prior to and after referral to an inpatient supportive care consult (SCC) for cancer pain management (CPM). Methods and Materials: Patients admitted at the MD Anderson Cancer Center and referred to a SCC were retrospectively reviewed. Cancer patients receiving SCH and BTO opioids for ≥24 h were eligible for inclusion. Patient demographics and clinical characteristics, including the type and route of SCH and BTO opioids, daily opioid doses (MEDDs) of SCH and BTO, and BTO/SCH ratios were reviewed in patients seen prior to a SCC (pre-SCC) and during a SCC. A normal BTO ratio was defined as 0.5-0.2. Results: A total of 665/728 (91%) patients were evaluable. Median pain scores (p < 0.001), BTO MEDDs (p < 0.001), scheduled opioid MEDDs (p < 0.0001), and total MEDDs (p < 0.0001) were higher, but the median number of BTO doses was fewer (2 vs. 4, p < 0.001), among patients seen at SCC compared to pre-SCC. A BTO/SCH ratio over the recommended ratio (>0.2) was seen in 37.5% of patients. The BTO/SCH ratios in the pre-SCC and SCC groups were 0.10 (0.04, 0.21) and 0.17 (0.10, 0.30), respectively, p < 0.001. Hydromorphone and Morphine were the most common BTO and SCH opioids prescribed, respectively. Patients in the early supportive care group had higher pain scores and MEDDs. Conclusions: BTO/SCH ratios are frequently prescribed higher than the recommended dose. Daily pain scores, BTO MEDDs, scheduled opioid MEDDs, and total MEDDs were higher among the SCC group than the pre-SCC group, but the number of BTO doses/day was lower.

Recent Advances in Fluorescence Recovery after Photobleaching for Decoupling Transport and Kinetics of Biomacromolecules in Cellular Physiology.

Among the new molecular tools available to scientists and engineers, some of the most useful include fluorescently tagged biomolecules. Tools, such as green fluorescence protein (GFP), have been applied to perform semi-quantitative studies on biological signal transduction and cellular structural dynamics involved in the physiology of healthy and disease states. Such studies focus on drug pharmacokinetics, receptor-mediated endocytosis, nuclear mechanobiology, viral infections, and cancer metastasis. In 1976, fluorescence recovery after photobleaching (FRAP), which involves the monitoring of fluorescence emission recovery within a photobleached spot, was developed. FRAP allowed investigators to probe two-dimensional (2D) diffusion of fluorescently-labelled biomolecules. Since then, FRAP has been refined through the advancements of optics, charged-coupled-device (CCD) cameras, confocal microscopes, and molecular probes. FRAP is now a highly quantitative tool used for transport and kinetic studies in the cytosol, organelles, and membrane of a cell. In this work, the authors intend to provide a review of recent advances in FRAP. The authors include epifluorescence spot FRAP, total internal reflection (TIR)/FRAP, and confocal microscope-based FRAP. The underlying mathematical models are also described. Finally, our understanding of coupled transport and kinetics as determined by FRAP will be discussed and the potential for future advances suggested.

Genomic islands of divergence infer a phenotypic landscape in Pacific lamprey.

High rates of dispersal can breakdown coadapted gene complexes. However, concentrated genomic architecture (i.e., genomic islands of divergence) can suppress recombination to allow evolution of local adaptations despite high gene flow. Pacific lamprey (Entosphenus tridentatus) is a highly dispersive anadromous fish. Observed trait diversity and evidence for genetic basis of traits suggests it may be locally adapted. We addressed whether concentrated genomic architecture could influence local adaptation for Pacific lamprey. Using two new whole genome assemblies and genotypes from 7,716 single nucleotide polymorphism (SNP) loci in 518 individuals from across the species range, we identified four genomic islands of divergence (on chromosomes 01, 02, 04, and 22). We determined robust phenotype-by-genotype relationships by testing multiple traits across geographic sites. These trait associations probably explain genomic divergence across the species' range. We genotyped a subset of 302 broadly distributed SNPs in 2,145 individuals for association testing for adult body size, sexual maturity, migration distance and timing, adult swimming ability, and larval growth. Body size traits were strongly associated with SNPs on chromosomes 02 and 04. Moderate associations also implicated SNPs on chromosome 01 as being associated with variation in female maturity. Finally, we used candidate SNPs to extrapolate a heterogeneous spatiotemporal distribution of these predicted phenotypes based on independent data sets of larval and adult collections. These maturity and body size results guide future elucidation of factors driving regional optimization of these traits for fitness. Pacific lamprey is culturally important and imperiled. This research addresses biological uncertainties that challenge restoration efforts.

Cold Atmospheric Plasma and Plasma-Activated Medium Trigger RONS-Based Tumor Cell Apoptosis.

The selective in vitro anti-tumor mechanisms of cold atmospheric plasma (CAP) and plasma-activated media (PAM) follow a sequential multi-step process. The first step involves the formation of primary singlet oxygen (1O2) through the complex interaction between NO2- and H2O2. 1O2 then inactivates some membrane-associated catalase molecules on at least a few tumor cells. With some molecules of their protective catalase inactivated, these tumor cells allow locally surviving cell-derived, extracellular H2O2 and ONOO─ to form secondary 1O2. These species continue to inactivate catalase on the originally triggered cells and on adjacent cells. At the site of inactivated catalase, cell-generated H2O2 enters the cell via aquaporins, depletes glutathione and thus abrogates the cell's protection towards lipid peroxidation. Optimal inactivation of catalase then allows efficient apoptosis induction through the HOCl signaling pathway that is finalized by lipid peroxidation. An identical CAP exposure did not result in apoptosis for nonmalignant cells. A key conclusion from these experiments is that tumor cell-generated RONS play the major role in inactivating protective catalase, depleting glutathione and establishing apoptosis-inducing RONS signaling. CAP or PAM exposure only trigger this response by initially inactivating a small percentage of protective membrane associated catalase molecules on tumor cells.

Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium.

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2-. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Persistent Dimer Emission in Thermally Activated Delayed Fluorescence Materials.

We expose significant changes in the emission color of carbazole-based thermally activated delayed fluorescence (TADF) emitters that arise from the presence of persistent dimer states in thin films and organic light-emitting diodes (OLEDs). Direct photoexcitation of this dimer state in 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) reveals the significant influence of dimer species on the color purity of its photoluminescence and electroluminescence. The dimer species is sensitive to the sample preparation method, and its enduring presence contributes to the widely reported concentration-mediated red shift in the photoluminescence and electroluminescence of evaporated thin films. This discovery has implications on the usability of these, and similar, molecules for OLEDs and explains disparate electroluminescence spectra presented in the literature for these compounds. The dimerization-controlled changes observed in the TADF process and photoluminescence efficiency mean that careful consideration of dimer states is imperative in the design of future TADF emitters and the interpretation of previously reported studies of carbazole-based TADF materials.

Stability of the Na+ Form of the Human Telomeric G-Quadruplex: Role of Adenines in Stabilizing G-Quadruplex Structure.

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature (Tm) was reduced from 59 °C for the wild-type sequence to 47 °C for the sequence where all four adenines were replaced with thymines (0123TTT). Furthermore, the enthalpy required for transitioning from the folded to unfolded G-quadruplex structure was reduced by 15 kcal/mol when the adenines were replaced with thymines (37 kcal/mol for the wild-type telomeric sequence reduced to 22 kcal/mol for the sequence where all four adenines were replaced with thymines (0123TTT)). The circular dichroism melting studies for G-quadruplex sequences having a single A to T change showed significantly sloping pretransition baselines and their differential scanning calorimetry (DSC) thermograms revealed biphasic melting profiles. In contrast, the deoxyoligonucleotides having sequences with two or more A to T changes did not exhibit sloping baselines or biphasic DSC thermograms. We attribute the biphasic unfolding profile and reduction in the enthalpy of unfolding to the energetic contributions of adenine hydrogen bonding within the loops as well as the adenine stacking to the G-tetrads of the G-quadruplex structure.

Frugal Biotech Applications of Low-Temperature Plasma.

Gas discharge low-temperature air plasma can be utilized for a variety of applications, including biomedical, at low cost. We term these applications 'frugal plasma' - an example of frugal innovation. We demonstrate how simple, robust, low-cost frugal plasma devices can be used to safely disinfect instruments, surfaces, and water.

Encapsulation and Ultrasound-Triggered Release of G-Quadruplex DNA in Multilayer Hydrogel Microcapsules.

Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy due to their intrinsic precision and selectivity for coding highly specific biological processes. However, freely administered nucleic acids of any type are quickly destroyed or rendered inert by a host of defense mechanisms in the body. In this work, we address the challenge of using nucleic acids as drugs by preparing stimuli responsive poly(methacrylic acid)/poly(N-vinylpyrrolidone) (PMAA/PVPON)n multilayer hydrogel capsules loaded with ~7 kDa G-quadruplex DNA. The capsules are shown to release their DNA cargo on demand in response to both enzymatic and ultrasound (US)-triggered degradation. The unique structure adopted by the G-quadruplex is essential to its biological function and we show that the controlled release from the microcapsules preserves the basket conformation of the oligonucleotide used in our studies. We also show that the (PMAA/PVPON) multilayer hydrogel capsules can encapsulate and release ~450 kDa double stranded DNA. The encapsulation and release approaches for both oligonucleotides in multilayer hydrogel microcapsules developed here can be applied to create methodologies for new therapeutic strategies involving the controlled delivery of sensitive biomolecules. Our study provides a promising methodology for the design of effective carriers for DNA vaccines and medicines for a wide range of immunotherapies, cancer therapy and/or tissue regeneration therapies in the future.

Regio- and conformational isomerization critical to design of efficient thermally-activated delayed fluorescence emitters.

Regio- and conformational isomerization are fundamental in chemistry, with profound effects upon physical properties, however their role in excited state properties is less developed. Here two regioisomers of bis(10H-phenothiazin-10-yl)dibenzo[b,d]thiophene-S,S-dioxide, a donor-acceptor-donor (D-A-D) thermally-activated delayed fluorescence (TADF) emitter, are studied. 2,8-bis(10H-phenothiazin-10-yl)dibenzo[b,d]thiophene-S,S-dioxide exhibits only one quasi-equatorial conformer on both donor sites, with charge-transfer (CT) emission close to the local triplet state leading to efficient TADF via spin-vibronic coupling. However, 3,7-bis(10H-phenothiazin-10-yl)dibenzo[b,d]thiophene-S,S-dioxide displays both a quasi-equatorial CT state and a higher-energy quasi-axial CT state. No TADF is observed in the quasi-axial CT emission. These two CT states link directly to the two folded conformers of phenothiazine. The presence of the low-lying local triplet state of the axial conformer also means that this quasi-axial CT is an effective loss pathway both photophysically and in devices. Importantly, donors or acceptors with more than one conformer have negative repercussions for TADF in organic light-emitting diodes.

Can stream and riparian restoration offset climate change impacts to salmon populations?

Understanding how stream temperature responds to restoration of riparian vegetation and channel morphology in context of future climate change is critical for prioritizing restoration actions and recovering imperiled salmon populations. We used a deterministic water temperature model to investigate potential thermal benefits of riparian reforestation and channel narrowing to Chinook Salmon populations in the Upper Grande Ronde River and Catherine Creek basins in Northeast Oregon, USA. A legacy of intensive land use practices in these basins has significantly reduced streamside vegetation and increased channel width across most of the stream network, resulting in water temperatures that far exceed the optimal range for salmon growth and survival. By combining restoration scenarios with climate change projections, we were able to evaluate whether future climate impacts could be offset by restoration actions. A combination of riparian restoration and channel narrowing was predicted to reduce peak summer water temperatures by 6.5 °C on average in the Upper Grande Ronde River and 3.0 °C in Catherine Creek in the absence of other perturbations. These results translated to increases in Chinook Salmon parr abundance of 590% and 67% respectively. Although projected climate change impacts on water temperature for the 2080s time period were substantial (i.e., median increase of 2.7 °C in the Upper Grande Ronde and 1.5 °C in Catherine Creek), we predicted that basin-wide restoration of riparian vegetation and channel width could offset these impacts, reducing peak summer water temperatures by about 3.5 °C in the Upper Grande Ronde and 1.8 °C in Catherine Creek. These results underscore the potential for riparian and stream channel restoration to mitigate climate change impacts to threatened salmon populations in the Pacific Northwest.

The Role of Local Triplet Excited States and D-A Relative Orientation in Thermally Activated Delayed Fluorescence: Photophysics and Devices.

Here, a comprehensive photophysical investigation of a the emitter molecule DPTZ-DBTO2, showing thermally activated delayed fluorescence (TADF), with near-orthogonal electron donor (D) and acceptor (A) units is reported. It is shown that DPTZ-DBTO2 has minimal singlet-triplet energy splitting due to its near-rigid molecular geometry. However, the electronic coupling between the local triplet (3LE) and the charge transfer states, singlet and triplet, (1CT, 3CT), and the effect of dynamic rocking of the D-A units about the orthogonal geometry are crucial for efficient TADF to be achieved. In solvents with low polarity, the guest emissive singlet 1CT state couples directly to the near-degenerate 3LE, efficiently harvesting the triplet states by a spin orbit coupling charge transfer mechanism (SOCT). However, in solvents with higher polarity the emissive CT state in DPTZ-DBTO2 shifts below (the static) 3LE, leading to decreased TADF efficiencies. The relatively large energy difference between the 1CT and 3LE states and the extremely low efficiency of the 1CT to 3CT hyperfine coupling is responsible for the reduction in TADF efficiency. Both the electronic coupling between 1CT and 3LE, and the (dynamic) orientation of the D-A units are thus critical elements that dictate reverse intersystem crossing processes and thus high efficiency in TADF.

Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.

Acute kidney injury (AKI) is one of the leading causes of in-hospital morbidity and mortality, particularly in critically ill patients. Although our understanding of AKI at the molecular level remains limited due to its complex pathophysiology, recent advances in both quantitative and spatial mass spectrometric approaches offer new opportunities to assess the significance of renal metabolomic changes in AKI models. In this study, we evaluated lipid changes in early ischemia-reperfusion (IR)-related AKI in mice by using sequential window acquisition of all theoretical spectra (SWATH)-mass spectrometry (MS) lipidomics. We found a significant increase in two abundant ether-linked phospholipids following IR at 6 h postinjury, a plasmanyl choline, phosphatidylcholine (PC) O-38:1 (O-18:0, 20:1), and a plasmalogen, phosphatidylethanolamine (PE) O-42:3 (O-20:1, 22:2). Both of these lipids correlated with the severity of AKI as measured by plasma creatinine. In addition to many more renal lipid changes associated with more severe AKI, PC O-38:1 elevations were maintained at 24 h post-IR, while renal PE O-42:3 levels decreased, as were all ether PEs detected by SWATH-MS at this later time point. To further assess the significance of this early increase in PC O-38:1, we used matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to determine that it occurred in proximal tubules, a region of the kidney that is most prone to IR injury and also rich in the rate-limiting enzymes involved in ether-linked phospholipid biosynthesis. Use of SWATH-MS lipidomics in conjunction with MALDI-IMS for lipid localization will help in elucidating the role of lipids in the pathobiology of AKI.

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

Tandem DART™ MS Methods for Methadone Analysis in Unprocessed Urine.

Current methods of methadone analysis in untreated urine are traditionally limited to enzyme immunoassays (EIA) while confirmation techniques require specimen processing (i.e., sample clean-up) before analyzing by gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS-MS). EIA and traditional confirmation techniques can be costly and, at times inefficient. As an alternative approach, we present Direct Analysis in Real Time (DART™) coupled with both time-of-flight and triple quadrupole linear ion trap (Q-TRAP™) mass spectrometers for screening and confirming methadone in untreated urine specimens. These approaches require neither expensive kits nor sample clean-up for analysis. More importantly, the total combined analysis time for both screening and confirmation methods was <5 min per sample; in contrast to the 3-5 day process required by traditional EIA, GC-MS and LC-MS-MS techniques. To examine the fundamental protocol and its applicability for routine drug screening, studies were performed that included limits of detection, precision, selectivity and specificity, sample recovery and stability and method robustness. The methods described in this report were determined to be highly specific and selective; allowing for detection of methadone at 250 ng/mL, consistent with cutoffs for current EIA techniques (300 ng/mL). The results reported here demonstrate the DART™ MS platform provides rapid and selective methadone analysis and the potential for providing savings of both time and resources compared with current analysis procedures.

Hypoxia regulates Toll-like receptor-9 expression and invasive function in human brain cancer cells in vitro.

Toll-like receptor-9 (TLR9) is a cellular DNA sensor of the innate immune system. TLR9 is widely expressed in a number of tumors, including brain cancer; however, little is known regarding its regulation and involvement in cancer pathophysiology. The present study demonstrated that hypoxia upregulates and downregulates TLR9 expression in human brain cancer cells in vitro, in a cell-specific manner. In addition, hypoxia-induced TLR9 upregulation was associated with hypoxia-induced invasion; however, such invasion was not detected in cells where hypoxia had suppressed TLR9 expression. Furthermore, suppression of TLR9 expression through TLR9 siRNA resulted in an upregulation of matrix metalloproteinase (MMP)-2, -9 and -13 and tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) mRNA, and a decreased invasion of cells in normoxia, in a cell-specific manner. In cells where hypoxia induced TLR9 expression, TLR9 expression and invasion were reduced by TLR9 siRNA. The decreased invasion observed in hypoxia was associated with the decreased expression of the MMPs and a concomitant increase in TIMP-3 expression. In conclusion, hypoxia regulates the invasion of brain cancer cells in vitro in a TLR9-dependent manner, which is considered to be associated with a complex expression pattern of TLR9-regulated mediators and inhibitors of invasion.

Recognition and binding of human telomeric G-quadruplex DNA by unfolding protein 1.

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1-G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na(+) and K(+) G-quadruplex-UP1 complexes (ΔH values of -43 and -19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na(+) form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10(8) M(-1) (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10(6) M(-1) (strand)]. Circular dichroism spectroscopy reveals the Na(+) form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.

Collective effects in vortex movements in complex plasmas.

We study the onset and characteristics of vortices in complex (dusty) plasmas using two-dimensional simulations in a setup modeled after the PK-3 Plus laboratory. A small number of microparticles initially self-arranges in a monolayer around the void. As additional particles are introduced, an extended system of vortices develops due to a nonzero curl of the plasma forces. We demonstrate a shear-thinning effect in the vortices. Velocity structure functions and the energy and enstrophy spectra show that vortex flow turbulence is present that is in essence of the "classical" Kolmogorov type.

Addition of bases to the 5'-end of human telomeric DNA: influences on thermal stability and energetics of unfolding.

Telomeric DNA has been intensely investigated for its role in chromosome protection, aging, cell death, and disease. In humans the telomeric tandem repeat (TTAGGG)n is found at the ends of chromosomes and provides a novel target for the development of new drugs in the treatment of age related diseases such as cancer. These telomeric sequences show slight sequence variations from species to species; however, each contains repeats of 3 to 4 guanines allowing the G-rich strands to fold into compact and stable nuclease resistant conformations referred to as G-quadruplexes. The focus of this manuscript is to examine the effects of 5'-nucleotides flanking the human telomeric core sequence 5'-AGGG(TTAGGG) 3-3' (h-Tel22). Our studies reveal that the addition of the 5'-flanking nucleotides (5'-T, and 5'-TT) results in significant changes to the thermodynamic stability of the G-quadruplex structure. Our data indicate that the observed changes in stability are associated with changes in the number of bound waters resulting from the addition of 5'-flanking nucleotides to the h-Tel22 sequence as well as possible intermolecular interactions of the 5' overhang with the core structure.